Constructs and Methods for Delivering Molecules via Viral Vectors with Blunted Innate Immune Responses

ABSTRACT

A CpG-modified recombinant adeno-associated viral (AAV) vector is described. The vector carries a nucleic acid molecule comprising AAV inverted terminal repeat (ITR) sequences and an exogenous gene sequence under the control of regulatory sequences which control expression of the gene product, in which the nucleic acid sequences carried by the vector are modified to significantly reduce CpG di-nucleotides such that an immune response to the vector is reduced as compared to the unmodified AAV vector. Also provided are methods and regimens for delivering transgenes using these AAV viral vectors, in which the innate immune response to the vector and/or transgene is significantly modulated.

STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support by 5-T32-HL-007954-12awarded by National Heart, Lung, and Blood Institute (NHLBI) and5-T32-AI-007324-20 awarded by the National Institutes of Health (NIH).The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV) is a small, non-enveloped human parvovirusthat packages a linear strand of single stranded DNA genome that is 4.7kb. The capsid of an AAV contains 60 copies (in total) of three viralproteins (VPs), VP1, VP2, and VP3, in a predicted ratio of 1:1:10,arranged with T=1 icosahedral symmetry [H-J Nam, et al., J Virol.,81(22): 12260-12271 (November 2007)]. The three VPs are translated fromthe same mRNA, with VP1 containing a unique N-terminal domain inaddition to the entire VP2 sequence at its C-terminal region [Nam etal., cited above]. VP2 contains an extra N-terminal sequence in additionto VP3 at its C terminus. In X-ray crystal structures of the AAV2 [Q.Xie, et al., Proc Natl. Acad. Sci. USA 99:10405-10410 (2002)] and AAV4[L. Govindasamy, et al., J. Virol., 80:11556-11570] capsids and allother structures determined for parvovirus capsids, only the C-terminalpolypeptide sequence in the AAV capsid proteins (˜530 amino acids) isobserved. The N-terminal unique region of VP1, the VP1-VP2 overlappingregion, and the first 14 to 16 N-terminal residues of VP3 are disordered[L. Govindasamy, et al., and Q. Xie et al., cited above].

Productive infection by AAV occurs only in the presence of a helpervirus, either adenovirus or herpes virus. In the absence of a helpervirus, AAV integrates into a specific point of the host genome (19q13-qter) at a high frequency, making AAV the only mammalian DNA virusknown to be capable of site-specific integration. See, Kotin et at.,1990, PNAS, 87: 2211-2215. However, recombinant AAV, which does notcontain any viral genes and only a therapeutic/marker expressioncassette packaged in an AAV capsid, does not integrate into the genome.Instead the recombinant viral genome fuses at its ends via invertedterminal repeats to form circular, episomal forms characterized by longterm gene expression.

The ability of AAV vectors to achieve long-term expression of thetransgene product has been attributed to their relatively lowimmunogenicity. However, in some experimental settings, attendant immuneresponses have compromised the outcome of AAV-mediated gene therapy. HowAAV activates the innate immune system remains unknown. In a studypublished by J. Zhu et al, J Clin Invest, Vol 119, No. 8 (August 2009),it is reported that the innate immune recognition of AAV2 byplasmacytoid dendric cells (DC) was mediated by TLR9 and dependent onMyD88. Activation of the TLR9-MyD88 pathway was independent of thenature of the transgene. Similarly, other serotypes of AAV, such as AAV1and AAV9, also activated innate immunity through the TLR9-MyD88 pathway.The authors conclude that their observations suggest that strategies toblock the TLR9-MyD88-type I IFN pathway may improve the clinical outcomeof AAV-mediated gene therapy.

There have been attempts to modulate the innate immune response to AAVvectors, which approaches have involved various methods of disruptingthe TLR9-MyD88-Type I IFN signaling pathway. One such approach isdescribed by Y. Yang et al, US Published Patent Application No.2011/0070241, published Mar. 24, 2011, which describes co-administrationof an antagonist of this pathway with the viral vector. Another approachis described in Yew et al, US Published Patent Application No.2012/0009222, published Jan. 12, 2012, which complexes lipidoids withpolynucleotides such as CpG oligonucleotides, in an attempt to modulateinnate immune responses. See, also, G. L. Rogers, et al, Frontiers inMicrobiology, Vol. 2, Article 194 (September 2011), a review articlewhich describes reducing vector load may be an alternative toartificially blocking the immune response with drugs. The authors ofthis paper also report on evidence that self-complementary (sc) AAVvectors induce a greater immune response than single-stranded (ss) AAV,indicating that there is some speculation this is perhaps due to a lackof stability of the viral capsid in scAAV vector. Rogers et al, furtherreport that the vector cassette does not affect the response and that itis unlikely specific sequences in the DNA are responsible [Rogers et al,cited above, page 8, spanning columns 1 and 2] for the response.

The approaches taken to date to address the innate immune responses toAAV have focused on the AAV capsid. In addition to attempts to modulateToll-like receptor 9 (TLR9) with short-term immunomodulators, e.g., TLR9antagonists, attempts have been made to make modifications to thecapsid, e.g., generating tyrosine mutant AAV capsids for AAV vectors toaddress these concerns.

What are needed in the art are constructs and methods for AAV-mediatedgene delivery that induce reduced or no detectable innate immuneresponses.

SUMMARY OF THE INVENTION

In one aspect, an AAV vector having a CpG-reduced or CpG-depletednucleic acid sequence packaged within an AAV capsid. Because the AAVvector is viral in origin and contains a capsid composed of proteins, nomodification to the viral capsid is necessary. A CpG-modified AAV vectorof the invention contains AAV inverted terminal repeat (ITR) sequencesand an exogenous gene sequence under the control of regulatory sequencesthat control expression of the gene product. The nucleic acid sequencesof one or more, and preferably all, of these elements are modified toreduce CpG di-nucleotides such that an immune response toward the vectorand/or transgene is reduced as compared to the unmodified (aka wildtype) AAV vector. Suitably, the AAV vector contains no other genomic AAVsequences.

In one embodiment, the transgene sequence contains a reduced number ofCpG di-nucleotides as compared to the native coding sequence for thegene product. In another embodiment, the regulatory sequences aremutated to reduce or eliminate CpG di-nucleotides. In still anotherembodiment, the 5′ and/or 3′ terminal repeat sequences are mutated toreduce or eliminate native CpG di-nucleotides.

In another aspect, the invention provides a composition comprising aCpG-modified DNA vector as described herein and a pharmaceuticallyacceptable carrier.

In a further aspect, the invention provides a method for improvingadeno-associated virus (AAV)-mediated gene expression by generating anAAV viral particle comprising a modified packaging insert, wherein saidpackaging insert comprises a nucleic acid molecule comprising AAVinverted terminal repeats (ITRs) sequences or functional equivalentsthereof, e.g., 5′ AAV ITR and 3′ AAV ITR (which ITRs may beindependently selected from CpG-modified or wild-type ITRs andoptionally CpG-modified self-complementary ITRs) and an exogenous genesequence under the control of regulatory sequences which controlexpression of the gene product, wherein said sequences of said nucleicacid molecule are modified to reduce CpG di-nucleotides such that animmune response to the vector is reduced as compared to the unmodifiedAAV vector without significant reduction in expression of the geneproduct; and delivering the AAV to a subject intramuscularly.

In still another aspect, the invention provides a regimen for repeatadministration of a gene product. The regimen comprises delivering to asubject an AAV vector having an AAV capsid having packaged therein aCpG-modified nucleic acid molecule carrying an exogenous gene sequenceand delivering to the subject a second vector comprising the exogenousgene sequence.

Still other aspects and advantages of the invention will be apparentfrom the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides representative sections of X-gal histochemical stain ofmuscle from wild-type (WT) and TLR9 knock-out (KO) mice injectedintramuscularly (IM) with 1×10¹¹ genome copies (GC) of AAVrh32.33nLacZ(nuclear LacZ). 4 mice/group.

FIG. 2A is a bar chart comparing nLacZ reactive CD8+ T cells in WT andTLR9KO mice on days 7, day 14, day 21, day 35, day 42 and day 60following intramuscular injection with 1×10¹¹ GC of AAVrh32.33nLacZ.Results represent the mean+/−SEM of tetramer positive orcytokine-producing cells from at least n=3 per group.

FIG. 2B is a bar chart showing gamma interferon (IFNγ) levels for bothwild-type and TLR9 knock out mice on days 35 and 60 followingintramuscular injection with 1×10¹¹ GC of AAVrh32.33nLacZ. Resultsrepresent the mean+/−SEM of cytokine-producing cells that are reactivetoward transgene (nLacZ) and AAVrh32.33 capsid antigen from at least n=3per group.

FIG. 3 provides photographs of representative muscle sections (4 miceper group) recovered from WT and TLR9KO mice that received I.M.injection of 1×10¹¹ GC of AAVrh32.33nLacZ. Sections were stained withanti-CD4 and anti-CD8 antibody (Ab) and examined by fluorescentmiscroscopy. 4 mice per group.

FIG. 4 shows photographs of X-gal histochemical stains of muscle from WTand TLR9KO mice injected intramuscularly with 1×10¹¹ GC ofAAVrh32.33nLacZ. Representative sections are from 35 and 60 dayspost-injection. 4 mice per group.

FIG. 5 provides photographs of muscle section that was recovered from WTand TLR9KO mice that received I.M. injection of either 1×10¹¹ GC ofAAVrh32.33 or AAV8 expressing nLacZ. Sections recovered on days 35 and60 were stained with anti-MHC II Ab and examined by fluorescentmiscroscopy. Representative sections are shown. 4 mice per group.

FIG. 6 provides a series of bar charts providing transcript levels ofvarious cytokines assessed by quantitative reverse transcriptase (RT)polymerase chain reaction (PCR) following isolation of RNA from thegastric muscle of WT and TLRKO mice administered 1×10¹¹ GCAAVrh32.33nLacZ or AAV8nLacZ at kinetic time points (2, 6, 12, 24hours). Transcript levels of MIP-1α, MIP-1β, IL-1β, IL-6, MIP-2 andMCP-1 were assessed by quantitative RT-PCR. Results depict the mean ofRNA expression. n=4 mice per group.

FIG. 7A illustrates the structure and sequence of the wild-type AAV2inverted terminal repeat showing AAV2 Rep binding site (RBS), spacersequence, terminal resolution site (trs), and rep binding element [SEQID NO:4]. FIG. 7B illustrates the AAV2 inverted terminal repeat with CpGdinucleotides outside of the RBS deleted [SEQ ID NO:5].

FIG. 8 provides photographs of representative sections of X-galhistochemical stains of muscle from WT mice injected I.M. with 1×10¹¹ GCof AAVrh32.33LacZCpG+ or of AAVrh32.33LacZCpG− vectors. Muscle washarvested at day 35 or day 60 post-injection. 4 mice per group.

FIG. 9A is a bar chart illustrating the percentage of LacZ reactive CD8+T cells in wild-type mice that were injected I.M. with 1×10¹¹ GC ofAAVrh32.33LacZCpG+ or CpG− vectors. Lymphocytes from the mice wereisolated from whole blood at various time points post-injection, i.e.,days 14, 21, 28, 42 and 60. Lymphocytes were subsequently stained usingthe PE-conjugated H-2K^(b)-ICPMYARV tetramer together with aFITC-conjugated anti-CD8 Ab to determine the percentage of LacZ-specificCD8+ T cells in the total CD8+ T cell population. Results represent themean+/−SEM of tetramer positive cells from at least n=3 recipients pergroup.

FIGS. 9B-9D are scattergrams illustrating the results of the sameexperiment as described in FIG. 9A. FIGS. 9B-9D provide a scattergramwhich illustrates the results of the study in the mice injected with theAAVrh32.33LacZCpG+ vectors (FIG. 9B), AAVrh32.33LacZCpG− vectors (FIG.9C) as compared to naïve animals (FIG. 9D), and which charts theconcentration of the βgal tetramer (PE-conjugated H-2K^(b)-ICPMYARVtetramer) versus the concentration CD8+ cells. FIG. 9E is a bar chartshowing gamma interferon (IFNγ) levels for wild-type mice on days 35 and60 following intramuscular injection with 1×10¹¹ GC ofAAVrh32.33LacZCpG+ or AAVrh32.33LacZCpG− vectors. Results represent themean+/−SEM of cytokine-producing cells that are reactive towardtransgene (nLacZ) and AAVrh32.33 capsid antigen from at least n=3 pergroup.

FIG. 10 provides photographs of representative muscle sections harvestedfrom WT mice that received I.M. injection of 1×10¹¹ GC ofAAVrh32.33nLacZCpG+ or CpG− vector. Sections from days 35 and 60following vector administration were stained with anti-CD4 and anti-CD8Ab and examined by fluorescent microscopy.

FIGS. 11A-11E provide the sequences of the wild-type (+CpG) [SEQ ID NO:6] and CpG-depleted (−CpG) [SEQ ID NO: 7] LacZ coding sequence in analignment which further provides a consensus sequence.

FIGS. 12A-12G provide the sequences of a CpG-modified human TnT intronwith S1001exons (−CpG) [SEQ ID NO: 8] in an alignment with the wild-typesequence (+CpG) [SEQ ID NO: 8] and a consensus sequence.

FIGS. 13A-13C provide the sequences of the wild-type (+CpG) [SEQ ID NO:10] and CpG-depleted (−CpG) firefly luciferase [SEQ ID NO: 11] codingsequence in an alignment which further provides a consensus sequence.

FIGS. 14A-14Q provide an alignment containing a CpG modified vector(pTJU28-ssCMV-S100A1+intron-CpG) with CpG depleted ITRs CMV-EF1-alphaenhancer/promoter S100A1 therapeutic transgene interrupted by a humantroponin T intron 9 modified to be CpG (−) followed by an atrialnatriuretic factor (ANF) 3′UTR and poly A+ modified to be CpG (−) [SEQID NO: 12] compared to the synthesized DNA fragment containing theS100A1+intron+ANF sequence [SEQ ID NO:13] then compared to the humantroponin T intron 9 sequence [SEQ ID NO: 14].

FIGS. 15A-15C provides diagrams of three pTJU vectors containing CpGdepleted ITRs, CpG (−) transgenes (LacZ, firefly luciferase and humanS100A1) and CpG (−) control elements.

FIG. 16 is a chart showing comparable LacZ plasmid expression for CpG+and CpG− AAV vector constructs. HeLa cells were transfected with CpG+and CpG− AAV expression plasmids. Four days post transfection cells wereassayed for β-galactosidase activity using the mammalian β-galactosidaseassay kit as instructed for adherent cells. Absorbance was measured at405 nm on a TECAN® Infinite M1000 PRO plate reader.

FIG. 17 is a chart showing significantly reduced Th1 responses in micethat receive RhCpG− vector on day 14 post administration. Splenocyteswere harvested 7 and 14 days intramuscular injection of RhCpG+ or RhCpG−vector. ELISPOT was performed to quantify spots of IFN-gamma per millioncells. Transgene reactive Th1 responses are similar between RhCpG+ andRhCpG− injected mice on day 7 but significantly reduced in RhCpG− genetransferred mice on day 14.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that there is a reduction in immuneresponse to a product expressed from a nucleic acid molecule expressioncassette carried by an AAV vector in which the expression cassette hasbeen CpG-modified to contain fewer CpG di-nucleotides. This contrastswith previous reports in the literature that AAV expression cassettesequences do not affect innate immune responses and this inventionprovides constructs and an approach which differs from that previouslytaken with AAV which involve a focus on the capsid involvement in theimmune response.

A CpG-modified adeno-associated viral (AAV) vector is described herein.As used herein, a cytosine monophosphate (C) followed by a guaninemonophosphate (G) in a nucleotide sequence is referred to as a CpGdinucleotide. In eukaryotes, the cytosine residues of CpG dinucleotidesare often methylated to 5-methyl-cytosine (mCpG). In bacterial genomes,CpG dinucleotides are typically unmethylated. As used throughout thisspecification, both methylated and unmethylated CpG are encompassed bythe use of the term “CpG” or “CpG dinucleotide), unless methylated onunmethylated CpG is specified.

In one embodiment, an AAV vector is a viral particle having an AAVcapsid in which a nucleic acid molecule is packaged. In one embodiment,the AAV vector lacks any functional AAV coding sequences, e.g., isdeleted in AAV rep and cap coding sequences. Preferably, the rep and capcoding regions are entirely deleted. Alternatively, these regions are atleast functionally deleted (i.e., incapable of producing rep or capproteins). To the extent any portion of the coding sequences areretained in the AAV vector, they are CpG-depleted and, preferably,CpG-free. Suitably, the nucleic acid molecule contains sequencesexogenous to the AAV capsid source, including a nucleic acid sequencewhich encodes a desired product for delivery to a selected host cell andregulatory sequences which direct expression thereof. The nucleic acidmolecule also contains two AAV ITRs, located 5′ and 3′ to the codingsequences, or functional equivalents to these AAV ITRs. These AAV ITRsmay be from the same source as the capsid, or may be from a differentAAV from the capsid which permits replication of the expression cassetteand packaging into the viral particle. Two AAV ITRs in a single vectormay also be from different sources from each other. Where the AAV ITRsare from a different source than the AAV capsid, the resulting viralvector particle is termed a pseudotyped AAV.

As previously described, the present invention does not require anymodifications to the AAV capsid or to the capsid coding sequence in theplasmid utilized for production of the AAV viral vector particle.Rather, it is the sequence carried within the AAV capsid which ismodified to reduce CpG di-nucleotides such that the immune response isreduced following delivery of the vector as compared to the responsegenerated by the unmodified AAV vector.

As used herein, the phrase “CpG− reduced” or “CpG-depleted” refers to anucleic acid sequence which is generated, either synthetically or bymutation of a nucleic acid sequence, such that a majority of the CpGdi-nucleotides are removed from the nucleic acid sequence. In someinstances, all CpG motifs are removed to provide what is termed hereinmodified CpG-free sequences. The CpG motifs are suitably reduced oreliminated not just in a coding sequence (e.g., a transgene), but alsoin the non-coding sequences, including, e.g., 5′ and 3′ untranslatedregions (UTRs), promoter, enhancer, polyA, ITRs, introns, and any othersequences present in the nucleic acid molecule. For the coding sequence,the DNA (5′ to 3′ direction) and codon triplets must be modified as wellas the interface between triplets.

For example, CpG di-nucleotides may be located within a codon tripletfor a selected amino acid. In one embodiment, the CpG di-nucleotidesallocated within a codon triplet for a selected amino acid is changed toa codon triplet for the same amino acid lacking a CpG di-nucleotide.

DNA Triplets DNA Triplets Amino Acid Containing CpG Lacking CpGSerine (Ser or S) TCG TCT, TCC, TCA, AGT, AGC Proline (Pro or P) CCGCCT, CCC, CCA, Threonine (Thr or T) ACG ACA, ACT, ACC Alanine (Ala or A)GCG GCT, GCC, GCA Arginine (Arg or R) CGT, CGC, AGA, AGG CGA, CGGAsparagine (Asn, N) AAT, AAC Aspartic acid (Asp, GAT, GAC D)Cysteine (Cys, C) TGT, TGC Glutamic acid (Glu, GAA, GAG E)Glutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGT, GGC, GGA, GGGHistidine (His, H) CAT, CAC Isoleucine (Ile, I) ATT, ATC, ATALeucine (Leu, L) CTT, CTC, ATA, CTG, TTA, TTG Lysine (Lys, K) AAA, AAGMethionine (Met, M) ATG Phenylalanine (Phe, TTT, TTC F) stopTAA, TAG, TGA Tryptophan (Trp, W) TGG Tyrosine (Tyr, Y) TAT, TACValine (Val, V) GTT, GTC, GTA, GTG

In addition, within the coding region, the interface between tripletsmust be taken into consideration. For example, if an amino acid tripletends in a C-nucleotide which is then followed by an amino acid tripletwhich can start only with a G-nucleotide (e.g., Valine, Glycine,Glutamic Acid, Alanine, Aspartic Acid), then the triplet for the firstamino acid triplet is changed to one which does not end in aC-nucleotide. Illustrative CpG-depleted coding sequences are illustratedin FIG. 12 for LacZ and FIG. 13 for luciferase.

Similarly, non-coding sequences carried within the AAV capsid are alsoaltered to be CpG-depleted and/or CpG-free.

Unless otherwise specified, the source of parental terminal repeats, AAVITRs, and other selected AAV components described herein, may be readilyselected from among any AAV, including, without limitation, AAV2, AAV7,AAV9 or another AAV sequences [e.g., US Published Patent Application No.2011-0263027 A1; US Published Patent Application No. US-2011-0236353A1,U.S. Pat. No. 7,282,199B1; WO 03/042397 A1; WO 2005/033321; WO2006/110689]. These parental ITRs or other AAV components may be readilyisolated from an AAV sequence using techniques available to those ofskill in the art of vector genome generation. Such parental AAV may beisolated or obtained from academic, commercial, or public sources (e.g.,the American Type Culture Collection, Manassas, Va.). Alternatively, theAAV sequences may be obtained through synthetic or other suitable meansby reference to published sequences such as are available in theliterature or in databases such as, e.g., GenBank®, PubMed®, or thelike. These parental AAV sequences are the AAV nucleic acid sequencesprior to CpG-depletion via synthetic methods or by site directedmutagenesis.

The wild-type ITRs from known AAV serotypes are CpG rich structures fromwhich CpG dinucleotides are reduced according to the invention withoutsignificantly impairing the ability of the ITRs to bind rep protein in apackaging host cell, as required in order to package an expressioncassette into an AAV viral vector. For example, the AAV2-5′ and 3′wild-type ITRs consist of 16 CpG dinucleotides (8 on each of the 5′ and3′ ITR). As illustrated herein, these ITRs can each be reduced to 8 CpGdinucleotides and still generate AAV vectors which transduce mousetissues. However, due to the location of the rep binding sequence on theAAV ITR, it has been found that these AAV2 ITRs may be difficult tomodify due to the CpG-free sequence affecting the rep-binding functionof the ITRs. Optionally, more CpG-dinucleotides may be reduced on theAAV2-3′ ITR or the AAV2 5′ ITRs, or combinations thereof, which do notaffect rep binding and vector packaging. Alternatively, ITRs from othernon-AAV2 sources may be selected which differ from AAV2 in the amount ofCpG di-nucleotides and the location of these motifs vis-à-vis the repbinding sequence, such as to permit the 5′ AAV ITR, the 3′ AAV ITR, orboth, to be rendered CpG-free according to the present invention.Similarly, modified ITRs, e.g., self-complementary ITR, or othersequences (e.g., parvovirus terminal repeats) which are functionallyequivalent to AAV 5′ ITRs and/or AAV 3′ ITRs can also be CpG-depletedand used in the present invention.

In addition to the ITRs, other regulatory sequences are desirablyCpG-depleted or rendered CpG-free according to the present invention.Such other regulatory sequences include a variety of elements including,e.g., without limitation, untranslated regions, promoter, enhancer,polyA, intron sequences, microRNAs and the like.

For example, the product encoded by the exogenous nucleic acid sequenceis typically under the control of a promoter and/or promoter/enhancersequence. Desirably, the CpG modifications to the promoters are made ina manner which does not affect the functional characteristics of thepromoter and/or enhancer, e.g., without affecting tissue preference.

In an embodiment, where it may not be possible to remove all CpGs from agiven nucleic molecule without negatively affecting a desired function,it may be desirable to concentrate on reducing clusters orconcentrations of CpG di-nucleotides. For example, promoter regions havebeen described as natively containing clusters of CpG di-nucleotides andthus have been used in the development of algorithms which can predictwhether removal of such CpG dinucleotides in this region will affectfunction in an unacceptable manner. Examples of such algorithms include,without limitation, those described in . . . Y. A. Medvedeva, et al,“Algorithms for CpG Islands Search: New Advantages and Old Problems, inBioinformatics—Trends and Methodologies”, p. 449-472 (2011); M.Hackenburg, et al, “Prediction of CpG-island function: CpG clusteringvs. sliding-window methods”, BMC Genomics, 26 May 2010, 11:327.Synthetic promoter design strategies can be employed to aid in reducingCpG without altering tissue preference.

Similar methods and algorithms can be used to predict which CpGmodifications may affect the function of a non-coding region. For othernon-coding sequences, however, e.g., an intron, this type of predictionis not required. For example, for an intron, the CpG modifications maybe performed so as to ensure that the altered nucleotides do not resultin an unwanted coding sequence.

Thus, in one embodiment, an AAV vector of the invention contains withinits capsid a nucleic acid sequence which contains a reduced number ofCpG di-nucleotides as compared to the sequences for the native elements.In one embodiment, the number of CpG di-nucleotides in the nucleic acidmolecule is reduced by at least about 25%, at least about 30%, at leastabout 45%, at least about 50%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or about 95%to 97% CpG-depleted as compared to a nucleic acid molecule having thecorresponding native sequences. One or more of the elements of thenucleic acid molecule carried by the AAV vector, e.g., withoutlimitation, the gene coding sequence, a promoter, an enhancer, anintron, the 3′ and 5′ UTRs, may be CpG-free, i.e., lacking any CpGdi-nucleotides.

In one embodiment, the immune response to the vector is reduced withoutsignificant reduction in expression of the gene product. Moreparticularly, in certain instances, changing codons to remove CpGdi-nucleotides may be result in less preferential codons being utilizedfor expression in a given type of host, resulting in lower expressionlevels of a transgene product. Suitably, codon triplets are selectedsuch that the immune response is reduced while retaining expressionlevels at a therapeutically or immunologically desired level.

In one embodiment, the CpG-depleted AAV vector retains about 100%, atleast about 95%, at least about 85%, at least about 80%, at least about75% of the protein expression levels as compared to a nucleic acidmolecule having the corresponding wild-type sequences, e.g., nativeITRs, native exogenous gene sequence and other regulatory sequences.Depending upon the amount of reduction of immune response, a moresignificant reduction in expression level may be found to be acceptablein order to achieve the therapeutic or immunologic expression levels.

Although to this point the specification has focused on AAV vectorswhich are CpG-depleted, the approach described herein may be applied toother DNA vectors, and particularly viral vectors which are enveloped orwhich have capsid proteins. Such viral vectors may include those basedon a double-stranded DNA virus, e.g., a virus selected from the groupconsisting of a baculovirus, poxvirus, herpesvirus or adenovirus, or asingle-stranded DNA virus, e.g., another member of the parvovirus familyof which AAV is a member.

The term “functional” refers to a product (e.g., a protein or peptide)which performs its native function, although not necessarily at the samelevel as the native product. The term “functional” may also refer to agene which encodes a product and from which a desired product can beexpressed. A “functional deletion” refers to a deletion which destroysthe ability of the product to perform its native function.

Unless otherwise specified (as above), the term fragments includespeptides at least 8 amino acids in length, at least 15 amino acids inlength, at least 25 amino acids in length. However, fragments of otherdesired lengths may be readily utilized depending upon the desiredcontext. Such fragments may be produced recombinantly or by othersuitable means, e.g., by chemical synthesis.

The term “percent (%) identity” may be readily determined for amino acidsequences, over the full-length of a protein, or a fragment thereof.Suitably, a fragment is at least about 8 amino acids in length, and maybe up to about 700 amino acids. Generally, when referring to “identity”,“homology”, or “similarity” between two different adeno-associatedviruses, “identity”, “homology” or “similarity” is determined inreference to “aligned” sequences. “Aligned” sequences or “alignments”refer to multiple nucleic acid sequences or protein (amino acids)sequences, often containing corrections for missing or additional basesor amino acids as compared to a reference sequence.

When alignments are referenced, or when the CpG-modified sequences arecompared to the sequences from the parental sequence prior tomodification, conventional alignment techniques may be utilized. Thereare a number of algorithms known in the art that can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta, a program in GCG Version 6.1. Fasta providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta with its default parameters (a word size of 6 and the NOPAM factorfor the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference. Similarly, programs are available forperforming amino acid alignments. Generally, these programs are used atdefault settings, although one skilled in the art can alter thesesettings as needed. Alternatively, one of skill in the art can utilizeanother algorithm or computer program that provides at least the levelof identity or alignment as that provided by the referenced algorithmsand programs.

Typically, when an alignment is prepared based upon an amino acidsequence (e.g., an AAV capsid protein), the alignment containsinsertions and deletions which are so identified with respect to areference AAV sequence (e.g., AAV2) and the numbering of the amino acidresidues is based upon a reference scale provided for the alignment.However, any given AAV sequence may have fewer amino acid residues thanthe reference scale. In the present invention, when discussing theparental sequence, the term “the same position” or the “correspondingposition” refers to the amino acid located at the same residue number ineach of the sequences, with respect to the reference scale for thealigned sequences. However, when taken out of the alignment, each of theproteins may have these amino acids located at different residuenumbers. Alignments are performed using any of a variety of publicly orcommercially available Multiple Sequence Alignment Programs. Sequencealignment programs are available for amino acid sequences, e.g., the“Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box”programs. Generally, any of these programs are used at default settings,although one of skill in the art can alter these settings as needed.Alternatively, one of skill in the art can utilize another algorithm orcomputer program which provides at least the level of identity oralignment as that provided by the referenced algorithms and programs.See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensivecomparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

As used throughout this specification and the claims, the terms“comprise” and “contain” and its variants including, “comprises”,“comprising”, “contains” and “containing”, among other variants, isinclusive of other components, elements, integers, steps and the like.The term “consists of” or “consisting of” are exclusive of othercomponents, elements, integers, steps and the like.

AAV Vectors with Expression Cassettes having CpG− Mutations

CpG-depleted AAV vectors are described as are methods for generatingthese sequences. More particularly, the sequence of any of the vectorelements described herein for CpG-depletion may be synthesized,generated via site-directed mutagenesis, or in some cases obtainedcommercially [e.g., CpG-free LacZ may be purchased from Invivogen(wild-type LacZ gene contains about 300 CpG di-nucleotides)]. Thetechniques by which these modifications are made is not a limitation onthe present invention. Similarly, once the CpG-depleted sequences areobtained, an AAV vector may be produced using any suitable method.

As previously described, the present invention does not requiremodifications to the AAV capsid (protein sequence) or to the sequenceused to produce same in a packaging host cell. A CpG-depleted AAV vectormay be generated according to the invention utilizing any functional AAVcapsid. In one embodiment, the capsid is provided by a single AAVsource. Suitable AAVs may be selected from among, e.g., AAV2, AAV8, andAAVrh32.22. Still other AAVs may be readily selected from amongst thosewhich have been described [US-2011-0263027A1; US-2011-0236353A1, U.S.Pat. No. 7,282,199B2; WO 03/042397 A1; WO 2005/033321; WO 2006/110689].Alternatively, the AAV capsid may be derived from more than one AAV.

Optionally, modified AAV capsid may be utilized to generate aCpG-depleted vector of the invention. For example, an AAV capsidcontaining a heparin binding site may be modified to ablate the heparinbinding motif, e.g., using methods such as those described in WO2008/027084. Still other modifications to the wild-type AAV capsid maybe made, e.g., to enhance production, yield and/or recovery of thecapsid [see, e.g., US Published Patent Application 2009-0197338A1describing “singleton” mutations] or an AAV vector having a chimeric orother artificial capsid protein. Other modifications include themodification(s) described in Yew et al, US Published Patent ApplicationNo. 2012/0009222, published Jan. 12, 2012.

In one aspect, the invention provides a method of generating aCpG-depleted adeno-associated virus (AAV) having an AAV capsid. Such amethod involves culturing a host cell which contains a nucleic acidsequence encoding an AAV capsid; a functional rep gene; and aCpG-depleted expression cassette for packaging into the AAV vector. Thecomponents required to be cultured in the host cell to package aCpG-modified expression cassette in an AAV capsid may be provided to thehost cell in trans. Alternatively, one or more of the requiredcomponents (e.g., expression cassette, rep sequences, cap sequences,and/or helper functions) may be provided by a stable host cell which hasbeen engineered to contain one or more of the required components usingmethods known to those of skill in the art. Most suitably, such a stablehost cell will contain the required component(s) under the control of aninducible promoter. However, the required component(s) may be under thecontrol of a constitutive promoter. Examples of suitable inducible andconstitutive promoters are provided herein, in the discussion ofregulatory elements suitable for use with the transgene. In stillanother alternative, a selected stable host cell may contain selectedcomponent(s) under the control of a constitutive promoter and otherselected component(s) under the control of one or more induciblepromoters. For example, a stable host cell may be generated which isderived from 293 cells (which contain E1 helper functions under thecontrol of a constitutive promoter), but which contains the rep and/orcap proteins under the control of inducible promoters. Still otherstable host cells may be generated by one of skill in the art.

The CpG-depleted expression cassette, rep sequences, cap sequences, andhelper functions required for producing the rAAV of the invention may bedelivered to the packaging host cell in the form of any genetic elementwhich transfer the sequences carried thereon. The selected geneticelement may be delivered by any suitable method, including thosedescribed herein. The methods used to construct any embodiment of thisinvention are known to those with skill in nucleic acid manipulation andinclude genetic engineering, recombinant engineering, and synthetictechniques. See, e.g., Sambrook et al, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly,methods of generating rAAV virions are well known and the selection of asuitable method is not a limitation on the present invention. See, e.g.,K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No.5,478,745.

Unless otherwise specified, the source of parental (i.e., wild-type orunmodified) AAV ITRs, and other selected AAV components describedherein, may be readily selected from among any AAV, including, thoseidentified herein. These ITRs or other AAV components may be readilyisolated using techniques available to those of skill in the art from anAAV sequence. Such AAV may be isolated or obtained from academic,commercial, or public sources (e.g., the American Type CultureCollection, Manassas, Va.). Alternatively, the AAV sequences may beobtained through synthetic or other suitable means by reference topublished sequences such as are available in the literature or indatabases such as, e.g., GenBank®, PubMed®, or the like. The AAV ITR andother expression cassette elements are CpG-depleted as described earlierin the specification.

While not repeated in each instance in the following description, itwill be readily understood that each of the expression cassette elementsmay be CpG-depleted according to the present invention and optionally,one or more of these elements (e.g., the coding sequence) may beCpG-free.

A. The CpG-Depleted Expression Cassette

The CpG-depleted expression cassette is composed of, at a minimum, atleast one copy of AAV inverted terminal repeat sequences, a transgeneand its regulatory sequences. In one embodiment, both 5′ AAV invertedterminal repeats (ITRs) and 3′ ITRs are included in the expressioncassette. In one desirable embodiment, the expression cassette containsITRs from an AAV sequence other than those which provided AAV capsidsequences. For example, pseudotyped vectors containing both 5′ and 3′CpG-modified AAV 2 ITRs may be used for convenience. However, a 5′ ITRand/or a 3′ ITR from other suitable AAVs may be selected and/orCpG-depleted as described herein. In another embodiment, functionalequivalents to one or both of the 5′ and 3′ AAV ITRs may be utilized.Such functional equivalents function as origins of DNA replication andas packaging signals for the viral genome to allow packaging of the DNAmolecule into the capsid to form a viral particle. For example, terminalrepeats from another parvovirus may provide a functional equivalent andserve as the source of parental terminal repeats (TRs) forCpG-depletion. The CpG-modified expression cassette is packaged into acapsid protein and delivered to a selected host cell.

1. The Transgene

The transgene is a nucleic acid sequence, exogenous to the AAV sequencesflanking the transgene and the source of the AAV capsid, which encodes apolypeptide, peptide, protein, enzyme, or other product of interest. Inone embodiment, the expression cassette carries a nucleic acid sequence,e.g., an RNA. Desirable RNA molecules include tRNA, dsRNA, RNAi,ribosomal RNA, catalytic RNAs, siRNA, small hairpin RNA, trans-splicingRNA, and antisense RNAs. One example of a useful RNA sequence is asequence which inhibits or extinguishes expression of a targeted nucleicacid sequence in the treated animal. Typically, suitable targetsequences include oncologic targets and viral diseases. This may beuseful, e.g., for cancer therapies and vaccines.

The nucleic acid coding sequence is suitably CpG-depleted as describedherein and operatively linked to regulatory components in a manner whichpermits transgene transcription, translation, and/or expression in ahost cell.

While any variety of products may be delivered using the constructs ofthe invention, the invention is particularly well suited for delivery ofproducts useful in diagnosis, treatment, and vaccination of conditionsassociated with conducting airway cells and amelioration of the symptomsthereof.

2. Regulatory Elements

In addition to the major elements identified above for the expressioncassette, the vector may also include conventional control elementswhich are CpG-depleted and which are operably linked to the transgene ina manner which permits its transcription, translation and/or expressionin a cell transfected with the plasmid vector or infected with the virusproduced by the invention. As used herein, “operably linked” sequencesinclude both expression control sequences that are contiguous with thegene of interest and expression control sequences that act in trans orat a distance to control the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [See, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the elongationfactor 1-alpha (EF1-alpha) promoter [Invitrogen]. Inducible promotersallow regulation of gene expression and can be regulated by exogenouslysupplied compounds, environmental factors such as temperature, or thepresence of a specific physiological state, e.g., acute phase, aparticular differentiation state of the cell, or in replicating cellsonly. Inducible promoters and inducible systems are available from avariety of commercial sources, including, without limitation, Invivogen,Invitrogen, Clontech and Ariad. Many other systems have been describedand can be readily selected by one of skill in the art. Examples ofinducible promoters regulated by exogenously supplied compounds,include, the zinc-inducible sheep metallothionine (MT) promoter, thedexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter,the T7 polymerase promoter system [International Patent Publication No.WO 98/10088]; the ecdysone insect promoter [No et al, Proc. Natl. Acad.Sci. USA, 93:3346-3351 (1996)], the tetracycline-repressible system[Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], thetetracycline-inducible system [Gossen et al, Science, 268:1766-1769(1995), See also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)], the RU486-inducible system [Wang et al, Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)] and therapamycin-inducible system [Magari et al, J. Clin. Invest.,100:2865-2872 (1997)]. Other types of inducible promoters which may beuseful in this context are those which are regulated by a specificphysiological state, e.g., temperature, acute phase, a particulardifferentiation state of the cell, or in replicating cells only.

In another embodiment, the native promoter for the transgene will beCpG-depleted and used. This promoter may be preferred when it is desiredthat expression of the transgene should mimic the native expression, orwhen expression of the transgene must be regulated temporally ordevelopmentally, in a tissue-specific manner, or in response to specifictranscriptional stimuli. In a further embodiment, other CpG-depletedexpression control elements, such as enhancer elements, polyadenylationsites or Kozak consensus sequences may also be used to mimic the nativeexpression.

Another embodiment of the transgene includes a CpG-depleted geneoperably linked to a tissue-specific promoter. For example, promotersspecific for pulmonary tissue and, where available, specific forconducting airway cells, may be used. Examples of such promoters mayinclude, e.g., the forkhead box J1 (FOXJ1) promoter, polyubiquitinpromoter UbC, SAM pointed domain-containing ETS transcription factor(SPDEF) promoter, Clara cell secretory protein/uteroglobin (CCSP/UG)promoter, amongst others.

Other lung-specific gene promoters may include, e.g., the surfactantprotein B (SPB), surfactant protein C (SPC), and surfactant protein A(SPA), ectogenic human carcinoembryonic antigen (CEA) promoter, Thyroidtranscription factor 1 (TTF1) and human surfactant protein A1 (hSPA1),amongst others.

Optionally, therapeutically useful transgenes may also includeselectable markers or reporter genes that include sequences encodinggeneticin, hygromicin or purimycin resistance, among others. Suchselectable reporters or marker genes (preferably located outside theviral genome to be rescued by the method of the invention) can be usedto signal the presence of the plasmids in bacterial cells, such asampicillin resistance. Other components of the plasmid may include anorigin of replication. Selection of these and other promoters and vectorelements are conventional and many such sequences are available.

The combination of the transgene, promoter/enhancer, and AAV ITRs isreferred to as an expression cassette for ease of reference herein.Having been provided with the teachings in this specification, thedesign of a CpG-depleted expression cassette can readily be made by oneof skill in the art.

3. Delivery of the CpG-Depleted Expression Cassette to a Packaging HostCell

The CpG-depleted expression cassette can be carried on any suitablevector, e.g., a plasmid, which is delivered to a packaging host cell.The plasmids useful in this invention may be engineered such that theyare suitable for replication and, optionally, integration in prokaryoticcells, mammalian cells, or both. These plasmids (or other vectorscarrying the 5′ AAV ITR-exogenous molecule-3′ AAV ITR) contain sequencespermitting replication of the expression cassette in eukaryotes and/orprokaryotes and selection markers for these systems. Selectable markersor reporter genes may include sequences encoding geneticin, hygromicinor purimycin resistance, among others. The plasmids may also containcertain selectable reporters or marker genes that can be used to signalthe presence of the vector in bacterial cells, such as ampicillinresistance. Other components of the plasmid may include an origin ofreplication and an amplicon, such as the amplicon system employing theEpstein Barr virus nuclear antigen. This amplicon system, or othersimilar amplicon components, permit high copy episomal replication inthe cells. Preferably, the molecule carrying the expression cassette istransfected into the cell, where it may exist transiently.Alternatively, the expression cassette (carrying the 5′ AAVITR-heterologous molecule-3′ ITR) may be stably integrated into thegenome of the host cell, either chromosomally or as an episome. Incertain embodiments, the expression cassette may be present in multiplecopies, optionally in head-to-head, head-to-tail, or tail-to-tailconcatamers. Suitable transfection techniques are known and may readilybe utilized to deliver the expression cassette to the host cell.

Generally, when delivering the vector comprising the CpG-depletedexpression cassette by transfection, the vector is delivered in anamount from about 5 μg to about 100 μg DNA, about 10 μg to about 50 μgDNA to about 1×10⁴ cells to about 1×10¹³ cells, or about 1×10⁵ cells.However, the relative amounts of vector DNA to host cells may beadjusted by one of ordinary skill in the art, who may take intoconsideration such factors as the selected vector, the delivery methodand the host cells selected.

B. Rep and Cap Sequences

In addition to the CpG-depleted expression cassette, the host cellcontains the sequences which drive expression of an AAV capsid in thehost cell and rep sequences of the same source as the source of the AAVITRs found in the CpG-depleted expression cassette, or across-complementing source. The AAV cap and rep sequences may beindependently obtained from an AAV source as described above and may beintroduced into the host cell in any manner known to one in the art asdescribed above. Additionally, when pseudotyping an AAV vector, thesequences encoding each of the essential rep proteins may be supplied bydifferent AAV sources (e.g., AAV2, AAV8, AAV32/33, AAV5, AAV7, AAV8,AAV9, or one of the other AAV sequences described herein or known in theart). For example, the rep78/68 sequences may be from AAV2, whereas therep52/40 sequences may be from AAV8.

In one embodiment, the host cell stably contains the capsid under thecontrol of a suitable promoter, such as those described above. Mostdesirably, in this embodiment, the capsid is expressed under the controlof an inducible promoter. In another embodiment, the capsid is suppliedto the host cell in trans. When delivered to the host cell in trans, thecapsid may be delivered via a plasmid that contains the sequencesnecessary to direct expression of the selected capsid in the host cell.Most desirably, when delivered to the host cell in trans, the plasmidcarrying the capsid also carries other sequences required for packagingthe rAAV, e.g., the rep sequences.

In another embodiment, the host cell stably contains the rep sequencesunder the control of a suitable promoter, such as those described above.Most desirably, in this embodiment, the essential rep proteins areexpressed under the control of an inducible promoter. In anotherembodiment, the rep proteins are supplied to the host cell in trans.When delivered to the host cell in trans, the rep proteins may bedelivered via a plasmid which contains the sequences necessary to directexpression of the selected rep proteins in the host cell.

Thus, in one embodiment, the rep and cap sequences may be transfectedinto the host cell on a single nucleic acid molecule and exist stably inthe cell as an episome. In another embodiment, the rep and cap sequencesare stably integrated into the chromosome of the cell. Anotherembodiment has the rep and cap sequences transiently expressed in thehost cell. For example, a useful nucleic acid molecule for suchtransfection comprises, from 5′ to 3′, a promoter, an optional spacerinterposed between the promoter and the start site of the rep genesequence, an AAV rep gene sequence, and an AAV cap gene sequence.

Optionally, the rep and/or cap sequences may be supplied on a vectorthat contains other DNA sequences that are to be introduced into thehost cells. For instance, the vector may contain the rAAV constructcomprising the expression cassette. The vector may comprise one or moreof the genes encoding the helper functions, e.g., the adenoviralproteins E1, E2a, and E4 ORF6, and the gene for VAI RNA.

Preferably, the promoter used in this construct may be any of theconstitutive, inducible or native promoters known to one of skill in theart or as discussed above. In one embodiment, an AAV P5 promotersequence is employed. The selection of the AAV to provide any of thesesequences does not limit the invention.

In another embodiment, the promoter for rep is an inducible promoter,such as are discussed above in connection with the transgene regulatoryelements. One preferred promoter for rep expression is the T7 promoter.The vector comprising the rep gene regulated by the T7 promoter and thecap gene, is transfected or transformed into a cell that eitherconstitutively or inducibly expresses the T7 polymerase. SeeInternational Patent Publication No. WO 98/10088, published Mar. 12,1998.

The spacer is an optional element in the design of the vector. Thespacer is a DNA sequence interposed between the promoter and the repgene ATG start site. The spacer may have any desired design; that is, itmay be a random sequence of nucleotides, or alternatively, it may encodea gene product, such as a marker gene. The spacer may contain geneswhich typically incorporate start/stop and polyA sites. The spacer maybe a non-coding DNA sequence from a prokaryote or eukaryote, arepetitive non-coding sequence, a coding sequence withouttranscriptional controls or a coding sequence with transcriptionalcontrols. Two exemplary sources of spacer sequences are the λ phageladder sequences or yeast ladder sequences, which are availablecommercially, e.g., from Gibco or Invitrogen, among others. The spacermay be of any size sufficient to reduce expression of the rep78 andrep68 gene products, leaving the rep52, rep40 and cap gene productsexpressed at normal levels. The length of the spacer may therefore rangefrom about 10 by to about 10.0 kbp, preferably in the range of about 100by to about 8.0 kbp. To reduce the possibility of recombination, thespacer is preferably less than 2 kbp in length; however, the inventionis not so limited.

Although the molecule(s) providing rep and cap may exist in the hostcell transiently (i.e., through transfection), it is preferred that oneor both of the rep and cap proteins and the promoter(s) controllingtheir expression be stably expressed in the host cell, e.g., as anepisome or by integration into the chromosome of the host cell. Themethods employed for constructing embodiments of this invention areconventional genetic engineering or recombinant engineering techniquessuch as those described in the references above. While thisspecification provides illustrative examples of specific constructs,using the information provided herein, one of skill in the art mayselect and design other suitable constructs, using a choice of spacers,P5 promoters, and other elements, including at least one translationalstart and stop signal, and the optional addition of polyadenylationsites.

In another embodiment of this invention, the rep or cap protein may beprovided stably by a host cell.

C. The Helper Functions

The packaging host cell also requires helper functions in order topackage the rAAV of the invention. Optionally, these functions may besupplied by a herpesvirus. Most desirably, the necessary helperfunctions are each provided from a human or non-human primate adenovirussource, such as those described above and/or are available from avariety of sources, including the American Type Culture Collection(ATCC), Manassas, Va. (US). In one currently preferred embodiment, thehost cell is provided with and/or contains an E1a gene product, an E1bgene product, an E2a gene product, and/or an E4 ORF6 gene product. Thehost cell may contain other adenoviral genes such as VAI RNA, but thesegenes are not required. In a preferred embodiment, no other adenovirusgenes or gene functions are present in the host cell.

The adenovirus E1a, E1b, E2a, and/or E4ORF6 gene products, as well asany other desired helper functions, can be provided using any means thatallows their expression in a cell. Each of the sequences encoding theseproducts may be on a separate vector, or one or more genes may be on thesame vector. The vector may be any vector known in the art or disclosedabove, including plasmids, cosmids and viruses. Introduction into thehost cell of the vector may be achieved by any means known in the art oras disclosed above, including transfection, infection, electroporation,liposome delivery, membrane fusion techniques, high velocity DNA-coatedpellets, viral infection and protoplast fusion, among others. One ormore of the adenoviral genes may be stably integrated into the genome ofthe host cell, stably expressed as episomes, or expressed transiently.The gene products may all be expressed transiently, on an episome orstably integrated, or some of the gene products may be expressed stablywhile others are expressed transiently. Furthermore, the promoters foreach of the adenoviral genes may be selected independently from aconstitutive promoter, an inducible promoter or a native adenoviralpromoter. The promoters may be regulated by a specific physiologicalstate of the organism or cell (i.e., by the differentiation state or inreplicating or quiescent cells) or by other means, e.g., by exogenouslyadded factors.

D. Host Cells And Packaging Cell Lines

The host cell itself may be selected from any biological organism,including prokaryotic (e.g., bacterial) cells, and eukaryotic cells,including, insect cells, yeast cells and mammalian cells. Particularlydesirable host cells are selected from among any mammalian species,including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2,BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, 293cells (which express functional adenoviral E1), Saos, C2C12, L cells,HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cellsderived from mammals including human, monkey, mouse, rat, rabbit, andhamster. The selection of the mammalian species providing the cells isnot a limitation of this invention; nor is the type of mammalian cell,i.e., fibroblast, hepatocyte, tumor cell, etc. The requirements for thecell used is that it not carry any adenovirus gene other than E1, E2aand/or E4 ORF6; it not contain any other virus gene which could resultin homologous recombination of a contaminating virus during theproduction of rAAV; and it is capable of infection or transfection ofDNA and expression of the transfected DNA. In a preferred embodiment,the host cell is one that has rep and cap stably transfected in thecell.

One host cell useful in the present invention is a host cell stablytransformed with the sequences encoding rep and cap, and which istransfected with the adenovirus E1, E2a, and E4ORF6 DNA and a constructcarrying the expression cassette as described above. Stable rep and/orcap expressing cell lines, such as B-50 (International PatentApplication Publication No. WO 99/15685), or those described in U.S.Pat. No. 5,658,785, may also be similarly employed. Another desirablehost cell contains the minimum adenoviral DNA which is sufficient toexpress E4 ORF6.

The preparation of a host cell according to this invention involvestechniques such as assembly of selected DNA sequences. This assembly maybe accomplished utilizing conventional techniques. Such techniquesinclude cDNA and genomic cloning, which are well known and are describedin Sambrook et al., cited above, use of overlapping oligonucleotidesequences of the adenovirus and AAV genomes, combined with polymerasechain reaction, synthetic methods, and any other suitable methods whichprovide the desired nucleotide sequence.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation, and/or infection by hybridadenovirus/AAV vectors into cell lines such as the human embryonickidney cell line HEK 293 (a human kidney cell line containing functionaladenovirus E1 genes which provides trans-acting E1 proteins).

Once generated, the CpG-depleted AAV vectors may be isolated andpurified using techniques known to those of skill in the art and used toprepare suitable formulation.

Pharmaceutical Compositions and Uses Therefor

These CpG-depleted AAV vectors may be prepared into a pharmaceuticalcomposition, typically in the form of a suspension with apharmaceutically acceptable carrier. The CpG-depleted AAV may bedelivered to host cells according to published methods. The AAV may beadministered to a human or non-human mammalian patient. Suitablecarriers may be readily selected by one of skill in the art in view ofthe indication for which the transfer virus is directed. For example,one suitable carrier includes saline, which may be formulated with avariety of buffering solutions (e.g., phosphate buffered saline). Otherexemplary carriers include sterile saline, lactose, sucrose, calciumphosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, andwater.

Optionally, the compositions may contain, in addition to the AAV andcarrier(s), other conventional pharmaceutical ingredients, such aspreservatives, or chemical stabilizers. Suitable exemplary preservativesinclude chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide,propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, andparachlorophenol. Suitable chemical stabilizers include gelatin andalbumin.

The vectors or AAV targeting moieties are administered in sufficientamounts to provide a therapeutic benefit without undue adverse effects,or with medically acceptable physiological effects, which can bedetermined by those skilled in the medical arts. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to a desired organ (e.g., the lung,liver, skeletal muscle, eye, heart), oral, inhalation, intranasal,intratracheal, intraarterial, intraocular, intravenous, intramuscular,subcutaneous, intradermal, and other parental routes of administration.Routes of administration may be combined, if desired.

Dosages of the viral vector or targeting moiety will depend primarily onfactors such as the condition being treated, the age, weight and healthof the patient, and may thus vary among patients. For example, atherapeutically effective human dosage of the viral vector is generallyin the range of from about 0.1 mL to about 100 mL of solution containingconcentrations of from about 1×10⁹ to 1×10¹⁶ genomes virus vector. Apreferred human dosage for delivery to large organs (e.g., liver,muscle, heart and lung) may be about 5×10¹⁰ to 5×10¹³ AAV genomes, at avolume of about 1 to 100 mL. A preferred dosage for delivery to eye isabout 5×10⁹ to 5×10¹² genome copies, at a volume of about 0.1 mL to 1mL. For example, a therapeutically effective human dosage of an airwayconducting cell targeting moiety is generally in the range of about 100μg to about 10 mg of the moiety. This may be delivered in solution,e.g., in about 0.1 mL to about 100 mL of solution. Optionally, dosageregimens similar to those described for therapeutic purposes may beutilized for immunization using the compositions of the invention. Forexample, an immunogically effective human dosage of the viral vector isgenerally in the range of from about 0.1 mL to about 100 mL of solutioncontaining concentrations of from about 1×10⁹ to 1×10¹⁶ genomes virusvector. One human dosage for delivery to large organs (e.g., liver,muscle, heart and lung) may be about 5×10¹⁰ to 5×10¹³ AAV genomes, at avolume of about 1 to 100 mL. One dosage for delivery to eye is about5×10⁹ to 5×10¹² genome copies, at a volume of about 0.1 mL to 1 mL.

In another embodiment, an amount of CpG-depleted AAV composition isadministered at an effective dose that is in the range of about 1×10⁸genome copies (GC) CpG-depleted AAV/kilogram (kg) to about 1×10¹⁴ GC/kg,and preferably 1×10¹¹ GC CpG-depleted AAV/kg to 1×10¹³ GC CpG-depletedAAV/kg to a human patient. Preferably, the amount of CpG-depleted viruscomposition administered is 1×10⁸ GC/kg, 5×10⁸ GC/kg, 1×10⁹ GC/kg, 5×10⁹GC/kg, 1×10¹⁰ GC/kg, 5×10¹⁰ GC/kg, 1×10¹¹ GC/kg, 5×10¹¹ GC/kg, or 1×10¹²GC/kg, 5×10¹² GC/kg, 1×10¹³ GC/kg, 5×10¹³ GC/kg, 1×10¹⁴ GC/kg.

These doses can be given once or repeatedly, such as daily, every otherday, weekly, biweekly, or monthly, or until adequate transgeneexpression is detected in the patient. In an embodiment, viruscompositions are given once weekly for a period of about 4-6 weeks, andthe mode or site of administration is preferably varied with eachadministration. Repeated injection is most likely required for completeablation of transgene expression. The same site may be repeated after agap of one or more injections. Also, split injections may be given.Thus, for example, half the dose may be given in one site and the otherhalf at another site on the same day.

Examples of therapeutic products and immunogenic products for deliveryby include those previously described for delivery via AAV constructsincluding those in, e.g., WO 2011/126808A2 and US Published PatentApplication No. 2009/0227030-A1, incorporated by reference herein. TheseCpG-depleted AAV vector may be used for a variety of therapeutic orvaccinal regimens, as described herein. Additionally, these CpG-depletedvectors may be delivered in combination with one or more other vectorsor active ingredients in a desired therapeutic and/or vaccinal regimen.For example, the CpG-depleted AAV compositions of the invention can beadministered to a human or non-human subject by any method described inthe following patents and patent applications that relate to methods ofusing AAV vectors in various therapeutic applications: U.S. Pat. Nos.7,282,199; 7,198,951; U.S. Patent Application Publication Nos. US2008-0075737; US 2008-0075740; International Patent ApplicationPublication Nos. WO 2003/024502; WO 2004/108922; WO 20051033321, each ofwhich is incorporated by reference in its entirety.

Treatment of Diseases and Disorders and Therapeutic Regimens

The invention provides methods for treating any disease or disorder thatis amenable to gene therapy. In one embodiment, “treatment” or“treating” refers to an amelioration of a disease or disorder, or atleast one discernible symptom thereof. In another embodiment,“treatment” or “treating” refers to an amelioration of at least onemeasurable physical parameter associated with a disease or disorder, notnecessarily discernible by the subject. In yet another embodiment,“treatment” or “treating” refers to inhibiting the progression of adisease or disorder, either physically, e.g., stabilization of adiscernible symptom, physiologically, e.g., stabilization of a physicalparameter, or both. Other conditions, including cancer, immunedisorders, and veterinary conditions, may also be treated.

Types of diseases and disorders that can be treated by methods of thepresent invention include, but are not limited to age-related maculardegeneration; diabetic retinopathy; infectious diseases e.g., HIV,pandemic flu, category 1 and 2 agents of biowarfare, or any new emergingviral infection; autoimmune diseases; cancer; multiple myeloma;diabetes; systemic lupus erythematosus (SLE); hepatitis C; multiplesclerosis; Alzheimer's disease; Parkinson's disease; amyotrophic lateralsclerosis (ALS), Huntington's disease; epilepsy; chronic obstructivepulmonary disease (COPD); joint inflammation, arthritis; myocardialinfarction (MI); congestive heart failure (CHF); hemophilia A; orhemophilia B.

Infectious diseases that can be treated or prevented by the methods ofthe present invention are caused by infectious agents including, but notlimited to, viruses, bacteria, fungi, protozoa, helminths, andparasites. The invention is not limited to treating or preventinginfectious diseases caused by intracellular pathogens. Many medicallyrelevant microorganisms have been described extensively in theliterature, e.g., See C. G. A Thomas, Medical Microbiology, BailliereTindall, Great Britain 1983, the entire contents of which are herebyincorporated herein by reference.

Therapeutic Transgenes

Useful therapeutic products encoded by the transgene include hormonesand growth and differentiation factors including, without limitation,insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH),growth hormone releasing factor (GRF), follicle stimulating hormone(FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG),vascular endothelial growth factor (VEGF), angiopoietins, angiostatin,granulocyte colony stimulating factor (GCSF), erythropoietin (EPO),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor(EGF), platelet-derived growth factor (PDGF), insulin growth factors Iand II (IGF-I and IGF-II), any one of the transforming growth factor αsuperfamily, including TGFα, activins, inhibins, or any of the bonemorphogenic proteins (BMP) BMPs 1-15 as well as TGFb proteins, any oneof the heregluin/neuregulin/ARIA/neu differentiation factor (NDF) familyof growth factors, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophicfactor (CNTF), glial cell line derived neurotrophic factor (GDNF),neurturin, agrin, any one of the family of semaphorins/collapsins,netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin,sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate theimmune system including, without limitation, cytokines and lymphokinessuch as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25(including IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractantprotein, leukemia inhibitory factor, granulocyte-macrophage colonystimulating factor, Fas ligand, tumor necrosis factors α and β,interferons α, β, TGFb and γ, stem cell factor, flk-2/flt3 ligand. Geneproducts produced by the immune system are also useful in the invention.These include, without limitations, immunoglobulins IgG, IgM, IgA, IgDand IgE, chimeric immunoglobulins, humanized antibodies, single chainantibodies, T cell receptors, chimeric T cell receptors, single chain Tcell receptors, class I and class II MHC molecules, as well asengineered immunoglobulins and MHC molecules. Useful gene products alsoinclude complement regulatory proteins such as complement regulatoryproteins, membrane cofactor protein (MCP), decay accelerating factor(DAF), CR1, CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation and/or lipid modulation, including the lowdensity lipoprotein (LDL) receptor, high density lipoprotein (HDL)receptor, the very low density lipoprotein (VLDL) receptor, andscavenger receptors. The invention also encompasses gene products suchas members of the steroid hormone receptor superfamily includingglucocorticoid receptors and estrogen receptors, Vitamin D receptors andother nuclear receptors. In addition, useful gene products includetranscription factors such as jun, fos, max, mad, serum response factor(SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins,TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1,CCAAT-box binding proteins, interferon regulation factor (IRF-1), Wilmstumor protein, ETS-binding protein, STAT, GATA-box binding proteins,e.g., GATA-3, and the forkhead family of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,cystathione beta-synthase, branched chain ketoacid decarboxylase,albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin cDNA sequence. Still other useful gene products includeenzymes such as may be useful in enzyme replacement therapy, which isuseful in a variety of conditions resulting from deficient activity ofenzyme. For example, enzymes that contain mannose-6-phosphate may beutilized in therapies for lysosomal storage diseases (e.g., a suitablegene includes that encoding β-glucuronidase (GUSB)).

Still other useful gene products include those used for treatment ofhemophilia, including hemophilia B (including Factor IX) and hemophiliaA (including Factor VIII and its variants, such as the light chain andheavy chain of the heterodimer and the B-deleted domain; U.S. Pat. No.6,200,560 and U.S. Pat. No. 6,221,349). The Factor VIII gene codes for2351 amino acids and the protein has six domains, designated from theamino to the terminal carboxy terminus as A1-A2-B-A3-C1-C2 [Wood et al,Nature, 312:330 (1984); Vehar et al., Nature 312:337 (1984); and Tooleet al, Nature, 342:337 (1984)]. Human Factor VIII is processed withinthe cell to yield a heterodimer primarily comprising a heavy chaincontaining the A1, A2 and B domains and a light chain containing the A3,C1 and C2 domains. Both the single chain polypeptide and the heterodimercirculate in the plasma as inactive precursors, until activated bythrombin cleavage between the A2 and B domains, which releases the Bdomain and results in a heavy chain consisting of the A1 and A2 domains.The B domain is deleted in the activated procoagulant form of theprotein. Additionally, in the native protein, two polypeptide chains(“a” and “b”), flanking the B domain, are bound to a divalent calciumcation.

In some embodiments, the minigene comprises first 57 base pairs of theFactor VIII heavy chain that encodes the 10 amino acid signal sequence,as well as the human growth hormone (hGH) polyadenylation sequence. Inalternative embodiments, the minigene further comprises the A1 and A2domains, as well as 5 amino acids from the N-terminus of the B domain,and/or 85 amino acids of the C-terminus of the B domain, as well as theA3, C1 and C2 domains. In yet other embodiments, the nucleic acidsencoding Factor VIII heavy chain and light chain are provided in asingle minigene separated by 42 nucleic acids coding for 14 amino acidsof the B domain [U.S. Pat. No. 6,200,560].

As used herein, a therapeutically effective amount is an amount of AAVvector that produces sufficient amounts of Factor VIII to decrease thetime it takes for a subject's blood to clot. Generally, severehemophiliacs having less than 1% of normal levels of Factor VIII have awhole blood clotting time of greater than 60 minutes as compared toapproximately 10 minutes for non-hemophiliacs.

The present invention is not limited to any specific Factor VIIIsequence. Many natural and recombinant forms of Factor VIII have beenisolated and generated. Examples of naturally occurring and recombinantforms of Factor VII can be found in the patent and scientific literatureincluding, U.S. Pat. No. 5,563,045, U.S. Pat. No. 5,451,521, U.S. Pat.No. 5,422,260, U.S. Pat. No. 5,004,803, U.S. Pat. No. 4,757,006, U.S.Pat. No. 5,661,008, U.S. Pat. No. 5,789,203, U.S. Pat. No. 5,681,746,U.S. Pat. No. 5,595,886, U.S. Pat. No. 5,045,455, U.S. Pat. No.5,668,108, U.S. Pat. No. 5,633,150, U.S. Pat. No. 5,693,499, U.S. Pat.No. 5,587,310, U.S. Pat. No. 5,171,844, U.S. Pat. No. 5,149,637, U.S.Pat. No. 5,112,950, U.S. Pat. No. 4,886,876, WO 94/11503, WO 87/07144,WO 92/16557, WO 91/09122, WO 97/03195, WO 96/21035, WO 91/07490, EP 0672 138, EP 0 270 618, EP 0 182 448, EP 0 162 067, EP 0 786 474, EP 0533 862, EP 0 506 757, EP 0 874 057, EP 0 795 021, EP 0 670 332, EP 0500 734, EP 0 232 112, EP 0 160 457, Sanberg et al., XXth Int. Congressof the World Fed. Of Hemophilia (1992), and Lind et al., Eur. J.Biochem., 232:19 (1995).

Nucleic acids sequences coding for the above-described Factor VIII canbe obtained using recombinant methods or by deriving the sequence from avector known to include the same. Furthermore, the desired sequence canbe isolated directly from cells and tissues containing the same, usingstandard techniques, such as phenol extraction and PCR of cDNA orgenomic DNA [See, e.g., Sambrook et al]. Nucleotide sequences can alsobe produced synthetically, rather than cloned. The complete sequence canbe assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence [See, e.g., Edge,Nature 292:757 (1981); Nambari et al, Science, 223:1299 (1984); and Jayet al, J. Biol. Chem. 259:6311 (1984).

Factor VIII from humans and non-human animals, including but not limitedto companion animals (e.g., canine, felines, and equines), livestock(e.g., bovines, caprines and ovines), laboratory animals, marinemammals, large cats, etc. are encompassed. The AAV vectors may contain anucleic acid coding for fragments of Factor VIII which is itself notbiologically active, yet when administered into the subject improves orrestores the blood clotting time. For example, as discussed above, theFactor VIII protein comprises two polypeptide chains: a heavy chain anda light chain separated by a B-domain which is cleaved duringprocessing. As demonstrated by the present invention, co-tranducingrecipient cells with the Factor VIII heavy and light chains leads to theexpression of biologically active Factor VIII. Because, however, mosthemophiliacs contain a mutation or deletion in only one of the chain(e.g., heavy or light chain), it may be possible to administer only thechain defective in the patient to supply the other chain.

Other useful gene products include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides having a non-naturally occurringamino acid sequence containing insertions, deletions or amino acidsubstitutions. For example, single-chain engineered immunoglobulinscould be useful in certain immunocompromised patients. Other types ofnon-naturally occurring gene sequences include antisense molecules andcatalytic nucleic acids, such as ribozymes, which could be used toreduce overexpression of a target.

Reduction and/or modulation of expression of a gene is particularlydesirable for treatment of hyperproliferative conditions characterizedby hyperproliferating cells, as are cancers and psoriasis. Targetpolypeptides include those polypeptides which are produced exclusivelyor at higher levels in hyperproliferative cells as compared to normalcells. Target antigens include polypeptides encoded by oncogenes such asmyb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu,trk and EGRF. In addition to oncogene products as target antigens,target polypeptides for anti-cancer treatments and protective regimensinclude variable regions of antibodies made by B cell lymphomas andvariable regions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune disease.Other tumor associated polypeptides can be used as target polypeptidessuch as polypeptides which are found at higher levels in tumor cellsincluding the polypeptide recognized by monoclonal antibody 17 lA andfolate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those whichmay be useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells which produce “self”-directed antibodies. Tcell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors (TCRs) and antibodies (Ab) that bindto endogenous antigens and initiate the inflammatory cascade associatedwith autoimmune diseases.

Immunogenic Transgenes

Suitably, the AAV vectors of the invention avoid the generation ofimmune responses to the AAV sequences contained within the vector.However, these vectors may nonetheless be formulated in a manner whichpermits the expression of a transgene carried by the vectors to inducean immune response to a selected antigen. For example, in order topromote an immune response, the transgene may be expressed from aconstitutive promoter, the vector can be adjuvanted as described herein,the transgene can optionally be modified to express more CpGs to inducea greater immune response, and/or the vector can be put intodegenerating tissue.

Examples of suitable immunogenic transgenes include those selected froma variety of viral families. Example of desirable viral families againstwhich an immune response would be desirable include, the picornavirusfamily, which includes the genera rhinoviruses, which are responsiblefor about 50% of cases of the common cold; the genera enteroviruses,which include polioviruses, coxsackieviruses, echoviruses, and humanenteroviruses such as hepatitis A virus; and the genera apthoviruses,which are responsible for foot and mouth diseases, primarily innon-human animals. Within the picornavirus family of viruses, targetantigens include the VP1, VP2, VP3, VP4, and VPG. Other viral familiesinclude the astroviruses and the calcivirus family. The calcivirusfamily encompasses the Norwalk group of viruses, which are an importantcausative agent of epidemic gastroenteritis. Still another viral familydesirable for use in targeting antigens for inducing immune responses inhumans and non-human animals is the togavirus family, which includes thegenera alphavirus, which include Sindbis viruses, RossRiver virus, andVenezuelan, Eastern & Western Equine encephalitis, and rubivirus,including Rubella virus. The flaviviridae family includes dengue, yellowfever, Japanese encephalitis, St. Louis encephalitis and tick borneencephalitis viruses. Other target antigens may be generated from theHepatitis C or the coronavirus family, which includes a number ofnon-human viruses such as infectious bronchitis virus (poultry), porcinetransmissible gastroenteric virus (pig), porcine hemagglutinatinencephalomyelitis virus (pig), feline infectious peritonitis virus(cats), feline enteric coronavirus (cat), canine coronavirus (dog), andhuman respiratory coronaviruses, which may cause the common cold and/ornon A, B or C hepatitis, and which include the putative cause of suddenacute respiratory syndrome (SARS). Within the coronavirus family, targetantigens include the E1 (also called M or matrix protein), E2 (alsocalled S or Spike protein), E3 (also called HE or hemagglutin elterose)glycoprotein (not present in all coronaviruses), or N (nucleocapsid).Still other antigens may be targeted against the arterivirus family andthe rhabdovirus family. The rhabdovirus family includes the generavesiculovirus (e.g., Vesicular Stomatitis Virus), and the generallyssavirus (e.g., rabies). Within the rhabdovirus family, suitableantigens may be derived from the G protein or the N protein. The familyfiloviridae, which includes hemorrhagic fever viruses such as Marburgand Ebola virus may be a suitable source of antigens. The paramyxovirusfamily includes parainfluenza Virus Type 1, parainfluenza Virus Type 3,bovine parainfluenza Virus Type 3, rubulavirus (mumps virus,parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastledisease virus (chickens), rinderpest, morbillivirus, which includesmeasles and canine distemper, and pneumovirus, which includesrespiratory syncytial virus. The influenza virus is classified withinthe family orthomyxovirus and is a suitable source of antigen (e.g., theHA protein, the N1 protein). The bunyavirus family includes the generabunyavirus (California encephalitis, La Crosse), phlebovirus (RiftValley Fever), hantavirus (puremala is a hemahagin fever virus),nairovirus (Nairobi sheep disease) and various unassigned bungaviruses.The arenavirus family provides a source of antigens against LCM andLassa fever virus. Another source of antigens is the bornavirus family.The reovirus family includes the genera reovirus, rotavirus (whichcauses acute gastroenteritis in children), orbiviruses, and cultivirus(Colorado Tick fever, Lebombo (humans), equine encephalosis, bluetongue). The retrovirus family includes the sub family oncorivirinalwhich encompasses such human and veterinary diseases as feline leukemiavirus, HTLVI and HTLVII, lentivirinal (which includes HIV, simianimmunodeficiency virus, feline immunodeficiency virus, equine infectiousanemia virus, and spumavirinal). The papovavirus family includes thesub-family polyomaviruses (BKU and JCU viruses) and the sub familypapillomavirus (associated with cancers or malignant progression ofpapilloma). The adenovirus family includes viruses (EX, AD7, ARD, O.B.)which cause respiratory disease and/or enteritis. The parvovirus familyfeline parvovirus (feline enteritis), feline panleucopeniavirus, canineparvovirus, and porcine parvovirus. The herpesvirus family includes thesub family alphaherpesvirinae, which encompasses the genera simplexvirus(HSVI, HSVII), varicellovirus (pseudorabies, varicella zoster) and thesub-family betaherpesvirinae, which includes the genera cytomegalovirus(HCMV, muromegalovirus) and the sub family gammaherpesvirinae, whichincludes the genera lymphocryptovirus, EBV (Burkitts lymphoma), humanherpesviruses 6A, 6B and 7, Kaposi's sarcoma-associated herpesvirus andcercopithecine herpesvirus (B virus), infectious rhinotracheitis,Marek's disease virus, and rhadinovirus. The poxvirus family includesthe sub family chordopoxvirinae, which encompasses the generaorthopoxvirus (Variola major (Smallpox) and Vaccinia (Cowpox)),parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus,and the sub family entomopoxvirinae. The hepadnavirus family includesthe Hepatitis B virus. One unclassified virus which may be suitablesource of antigens is the Hepatitis delta virus, Hepatitis E virus, andprions. Another virus which is a source of antigens is Nipan Virus.Still other viral sources may include avian infectious bursal diseasevirus and porcine respiratory and reproductive syndrome virus. Thealphavirus family includes equine arteritis virus and variousEncephalitis viruses.

The present invention may also encompass immunogens which are useful toimmunize a human or non-human animal against other pathogens includingbacteria, fungi, parasitic microorganisms or multicellular parasiteswhich infect human and non-human vertebrates, or from a cancer cell ortumor cell. Examples of bacterial pathogens include pathogenic grampositive cocci include pneumococci; staphylococci (and the toxinsproduced thereby, e.g., enterotoxin B); and streptococci. Pathogenicgram negative cocci include meningococcus; gonococcus. Pathogenicenteric gram negative bacilli include enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigella;haemophilus; moraxella; H. ducreyi (which causes chancroid); brucellaspecies (brucellosis); Francisella tularensis (which causes tularemia);Yersinia pestis (plague) and other yersinia (pasteurella);streptobacillus moniliformis and spirillum; Gram-positive bacilliinclude listeria monocytogenes; erysipelothrix rhusiopathiae;Corynebacterium diphtheria (diphtheria); cholera; B. anthracis(anthrax); donovanosis (granuloma inguinale); and bartonellosis.Diseases caused by pathogenic anaerobic bacteria include tetanus;botulism (Clostridum botulinum and its toxin); Clostridium perfringensand its epsilon toxin; other clostridia; tuberculosis; leprosy; andother mycobacteria. Pathogenic spirochetal diseases include syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude glanders (Burkholderia mallei); actinomycosis; nocardiosis;cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis;candidiasis, aspergillosis, and mucormycosis; sporotrichosis;paracoccidiodomycosis, petriellidiosis, torulopsosis, mycetoma andchromomycosis; and dermatophytosis. Rickettsial infections includeTyphus fever, Rocky Mountain spotted fever, Q fever (Coxiella burnetti),and Rickettsialpox. Examples of mycoplasma and chlamydial infectionsinclude: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis;and perinatal chlamydial infections. Pathogenic eukaryotes encompasspathogenic protozoans and helminths and infections produced therebyinclude: amebiasis; malaria; leishmaniasis; trypanosomiasis;toxoplasmosis; Pneumocystis carinii; Trichans; Toxoplasma gondii;babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis;nematodes; trematodes or flukes; and cestode (tapeworm) infections.

Many of these organisms and/or the toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHealth and Human Services, USA], as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracis (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fevers[filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa,Machupo]), all of which are currently classified as Category A agents;Coxiella burnetti (Q fever); Brucella species (brucellosis),Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis),Ricinus communis and its toxin (ricin toxin), Clostridium perfringensand its toxin (epsilon toxin), Staphylococcus species and their toxins(enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats(e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsiapowazekii), and viral encephalitis (alphaviruses, e.g., Venezuelanequine encephalitis; eastern equine encephalitis; western equineencephalitis); all of which are currently classified as Category Bagents; and Nipan virus and hantaviruses, which are currently classifiedas Category C agents. In addition, other organisms, which are soclassified or differently classified, may be identified and/or used forsuch a purpose in the future. It will be readily understood that theviral vectors and other constructs described herein are useful todeliver antigens from these organisms, viruses, their toxins or otherby-products, which will prevent and/or treat infection or other adversereactions with these biological agents.

The vectors of the invention can be used to deliver immunogens. Inrheumatoid arthritis (RA), several specific variable regions of T-cellreceptors (TCRs) which are involved in the disease have beencharacterized. These TCRs include V 3, V 14, V 17 and V 17. Thus,delivery of a nucleic acid sequence that encodes at least one of thesepolypeptides will elicit an immune response that will target T cellsinvolved in RA. In multiple sclerosis (MS), several specific variableregions of TCRs which are involved in the disease have beencharacterized. These TCRs include V 7 and V 10. Thus, delivery of anucleic acid sequence that encodes at least one of these polypeptideswill elicit an immune response that will target T cells involved in MS.In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs include V 6,V 8, V 14 and V 16, V 3C, V 7, V 14, V 15, V 16, V 28 and V 12. Thus,delivery of a nucleic acid molecule that encodes at least one of thesepolypeptides will elicit an immune response that will target T cellsinvolved in scleroderma.

A CpG-modified AAV viral vector of the invention provides an efficientgene transfer vehicle which can deliver a selected transgene to aselected host cell in vivo or ex vivo even where the organism hasneutralizing antibodies to one or more AAV serotypes. In one embodiment,the rAAV and the cells are mixed ex vivo; the infected cells arecultured using conventional methodologies; and the transduced cells arere-infused into the patient.

These compositions are particularly well suited to gene delivery fortherapeutic purposes and for immunization, including inducing protectiveimmunity. Further, the compositions of the invention may also be usedfor production of a desired gene product in vitro. For in vitroproduction, a desired product (e.g., a protein) may be obtained from adesired culture following transfection of host cells with a rAAVcontaining the molecule encoding the desired product and culturing thecell culture under conditions which permit expression. The expressedproduct may then be purified and isolated, as desired. Suitabletechniques for transfection, cell culturing, purification, and isolationare known to those of skill in the art.

In one embodiment, a method for improving adeno-associated virus(AAV)-mediated gene expression is described. The method involvesgenerating an AAV viral particle comprising a modified packaging insert,wherein said packaging insert comprises a nucleic acid moleculecomprising at least one AAV inverted terminal repeat (ITR) and anexogenous gene sequence under the control of regulatory sequences whichcontrol expression of the gene product, wherein said sequences of saidnucleic acid molecule are modified to reduce CpG di-nucleotides suchthat in immune response to the vector is reduced as compared to theunmodified AAV vector without significant reduction in expression of thegene product; and delivering the AAV to a subject intramuscularly. Inanother embodiment, a regimen for repeat administration of a geneproduct. The regimen involves delivering to a subject a CpG-depletedadeno-associated viral (AAV) vector. The vector has an AAV capsid havingpackaged therein a nucleic acid molecule comprising at least one AAVinverted terminal repeat (ITR) and an exogenous gene sequence under thecontrol of regulatory sequences which control expression of the geneproduct, wherein said sequences of said nucleic acid molecule aremodified to reduce CpG di-nucleotides such that in immune response tothe vector is reduced as compared to the unmodified AAV vector withoutsignificant reduction in expression of the gene product; and deliveringto the subject a second vector comprising the exogenous gene sequence.The second vector may be a second CpG-depleted AAV, which may differfrom the first CpG-depleted AAV. Alternatively, a CpG-depleted AAV asdescribed herein may be used in a regimen using other types of AAVvector, or other viral and non-viral constructs. For example, regimensanalogous to those described in EP 1 742 668B1 and WO 2006/078279A2,published 27 Jul. 2006, may be performed using a CpG-depleted AAV of theinvention.

EXAMPLES

The following examples are illustrative only and are not a limitation onthe present invention.

Example 1 In the Absence of TLR9 Signaling, AAVrh32.33nLacZ Muscle GeneTransfer Results in Stable Transgene Expression

The current study assessed the requirement for TLR9 signaling in T cellimmunoreactivity and transgene loss in response to AAVrh32.33. Thesemechanistic findings were subsequently translated into a modified,CpG-depleted AAVrh32.33 vector that escapes the adaptive immune responseand exhibits stable, long-term transgene expression.

A. Material and Methods:

1. Mice: C57BL/6 wild type (WT) mice were ordered from The JacksonLaboratory. Toll-like receptor 9 knockout (TLR9KO) mice were a kind giftfrom Dr. Phillip Scott (University of Pennsylvania, Philadelphia, Pa.).All mice were housed under specific pathogen-free conditions in the TRLAnimal Facility at the University of Pennsylvania. All animal procedureprotocols were approved by the Institutional Animal Care and UseCommittee of the University of Pennsylvania.

2. Adeno-Associated Viral Vectors (AAV) mediated transduction of themuscle

An AAV8 and AAVrh32.33 pseudotyped vector flanked with AAV2 ITRs encodeda nuclear targeted form of β-galactosidase (nLacZ) under thetranscriptional control of a CMV-enhanced chicken β-actin (CB) promoter[L. Wang, et al., 1999. Sustained correction of bleeding disorder inhemophilia B mice by gene therapy. Proc Natl Acad Sci USA 96:3906-3910.] An AAVrh32.33 pseudotyped vector flanked with AAV2 ITRsencoded either a wild type or CpG-depleted cellular targeted form ofβ-galactosidase (LacZ) (Invivogen). The CpG-depleted promoter and polyAfor this construct were also obtained from Invivogen. Over 320 CpGs arepresent in the wild type LacZ expressing vector (16 CpGs in the invertedterminal repeats (ITRs) and 308 in the transgene); the CpG depleted LacZvector sequence contains only 16 CpGs located in the ITRs. Outside ofthe transgene sequence, both vectors are identical in sequence andcontain CpG-depleted human elongation factor 1-alpha (E1F-α) promoter,CMV enhancer, intron, SV40 3′UTR, and ITRs. AAV vectors were produced bya scaled-down version of a previously described method by tripletransfection of vector genome, AAV helper and adenovirus helper plasmids[M. Lock, et al, 2010. Rapid, simple and versatile manufacturing ofrecombinant adeno-associated viral vectors at scale. Hum Gene Ther 21:1259-1271]. Purification of vectors involved a single iodixanol stepgradient [Zolotukhin, S., et al., 1999]. Recombinant adeno-associatedvirus purification using novel methods improves infectious titer andyield. Gene Ther 6: 973-985] and subsequent DNAse treatment. Real-timePCR using a primer/probe set corresponding to the poly A region of thevector and linearized plasmid standards determined genome titer (genomecopy/ml) of AAV vectors. All vectors used in this study passed theendotoxin assay (threshold 10 EU/mL) using QCL-1000 chromogenic LAL testkit (Cambrex Bio Science). Vectors were produced by Penn Vector Core atthe University of Pennsylvania. Mice were injected in the gastroc with10¹¹ vector genomes (VG) of AAV in a 50 μl volume.

3. Immunohistochemistry

To examine expression of nuclear β-gal, X-gal staining of snap-frozenliver cryosections was performed according to standard protocols [Bell,P., et al., Histochem Cell Biol 124: 77-85.] Representative sectionsfrom 4 mice per group were imaged by using brightfield microscopy with a10× objective. To analyze major histocompatibility complex class II (MHCII) expression and CD4+/CD8+ infiltrating cell types within the liver,immunostaining and fluorescent microscopy were performed onacetone-fixed cryosections stained with rat anti-CD4 and anti-CD8antibodies (Ab) from BD Pharmingen and anti-MHC II Ab (Biolegend) aspreviously described [Mays, L. E., and J. M. Wilson. 2009. J Gene Med11:1095-1102].

4. MHC Class I Tetramer Staining

PE-conjugated MHC class I H2-k^(b)-ICPMYARV tetramer complex wasobtained from Beckman Coulter. At kinetic time points after vectorinjection, tetramer staining was performed on heparinized whole bloodcells isolated by retro-orbital bleeds. Cells were co-stained for 30minutes at room temperature with PE-conjugated tetramer andFITC-conjugated anti-CD8a (Ly-2) Ab (BD Pharmingen). Red blood cellswere lysed and cells were fixed with iTAg MHC tetramer lysing solutionsupplemented with fix solution (Beckman Coulter) for 15 minutes at roomtemperature. The cells were then washed three times in PBS andresuspended in 1×PBS. Data were gathered with an FC500 Flow Cytometer(Beckman Coulter) and were analyzed with FlowJo analysis software (TreeStar). In the analysis, lymphocytes were selected on the basis offorward and side scatter characteristics, followed by selection of CD8+cells, and subsequently the tetramer-positive CD8+ T cell population.

5. ELISPOT Assays for Cytokine-Producing Cells

T cell medium consisted of the following: DMEM (Cellgro; Mediatech)supplemented with 10% heat-inactivated FBS (Hyclone), 1%penicillin/streptomycin (Cellgro; Mediatech), 1% L-glutamine (Cellgro;Mediatech), 10 mM HEPES buffer (Cellgro; Mediatech), 0.1 mM nonessentialamino acids (Invitrogen), 2 mM sodium pyruvate Cellgro; Mediatech) and10⁻⁶ M 2-ME (Cellgro; Mediatech).

Splenocytes were isolated by mechanical dissociation followed by redblood cell (RBC) lysis via hypotonic shock and resuspended at aconcentration of 5×10⁶ cells/mL and plated at 5×10⁵ cells/well intriplicate on 96-well round-bottom plates. ELISPOT assays were performedaccording to the manufacturer's instructions (BD Biosciences). T cellmedium supplemented with 2 μg/mL of H2-k^(b)-restricted β-gal CD8 T cellepitope (ICPMYARV) and H2-k^(b)-restricted AAVrh32.33 capsid epitope(SSYELPYVM) (Mimotopes) was used to stimulate splenocytes. Splenocyteswere incubated at 37° C., 5% CO₂ for 18 hours. Spots were visualized byaddition of 3-Amino-9-Ethylcarbazole (AEC) substrate set (BDBiosciences) and counted using the AID ELISPOT reader system (CellTechnology).

6. RNA Isolation and Quantitative RT-PCR

Gastroc tissue was homogenized in 1 ml TRIzol (Life Technologies) andRNA was isolated per the manufacturer's protocol. Total RNA (5 μg) wasreverse transcribed using 10×PCR buffer (Roche), 10 mM dNTP, oligo(dt),M-MLV-RT (all from Invitrogen), and RNAsin (Promega). Products were thencleaned with 1:1 phenol/chloroform/isoamyl (25:24:1) and reprecipitatedwith 7.5 M NH₄OAC in pure ethanol overnight at −80° C.

Real-time PCR was performed on cDNA using a 7500 Real Time PCR System(Applied Biosystems). Primer binding to DNA was detected by SYBR 2XMaster Mix (Applied Biosystems). Relative expression of the gene ofinterest was expressed as the comparative concentration of the geneproduct to the GAPDH product. Transcript relative expression of targetgenes were then expressed as fold induction over mock treated (PBSinjected) naïve WT mouse gastroc samples. Significance was determinedwith an unpaired Student t test.

7. Muscle Weight

Muscle weight was determined by weighing the vector injected gastroc andcomparing it to the total body weight of the animal on day 60post-injection.

8. Statistical Analysis

Data were analyzed with GraphPad Prism 4.0c software using unpairedStudent t-tests. p values of ≦0.05 were considered statisticallysignificant.

Results:

B. In the Absence of TLR9 Signaling, AAVrh32.33nLacZ Muscle GeneTransfer Results in Stable Transgene Expression.

Studies in C57BL/6 mice demonstrate that AAVrh32.33 intramuscular genetransfer induces a robust adaptive immune response towards both capsidand transgene antigen, heavy cellular infiltrate, and a loss ofdetectable transgene expression [Mays, L. E., et al, 2009. J of Immunol182: 6051-6060]. To evaluate the role of TLR9 signaling in the inductionof this adaptive immune response and transgene loss, WT and TLR9KO micewere intramuscularly (I.M.) injected with 1×10¹¹ viral particles ofAAVrh32.33 expressing a nuclear-targeted βgal (nLacZ) reporter geneunder the direction of a chicken β-actin promoter. Gastroc tissue wasrecovered from the WT and TLR9KO mice and βgal expression in the musclewas assessed by X-gal histochemical stain (FIG. 1). WT controlsexhibited a complete loss of β-gal positive cells at 60 dayspost-injection. In contrast, abrogation of TLR9 signaling resulted instable transgene expression. These results suggest that TLR9 signalingis required for transgene loss following AAVrh32.33 muscle genetransfer.

C. A Deficiency in TLR9 Signaling Reduces Immunoreactivity TowardAAVrh32.33 Capsid and Transgene Antigen.

In WT C57BL/6 mice, AAVrh32.33 muscle gene transfer is associated with arobust Th1 response and a significant percentage of nLacZ reactive CD8+T cells [Mays et al, 2009, J Immunol, cited above]. To investigate therelationship between TLR9 signaling and immunoreactivity, MHC I tetramerstain and ELISPOT assays were used to quantify transgene reactive CD8+ Tcells and primed transgene and capsid responsive IFNγ-producing cells(FIG. 2). Peripheral blood cells isolated from whole blood wereco-stained with a FITC-conjugated anti-CD8 Ab and a PE-conjugatedH-2K^(b)-ICPMYARV tetramer to determine the percentage of nLacZ-reactiveCD8+ T cells in the total CD8+ T cell population (FIG. 2A). TLR9deficient mice exhibited a significant reduction in the percentage ofnLacZ responsive CD8+ T cell population compared to WT mice.

To determine whether TLR9 signaling regulates AAVrh32.33nLacZ cellularimmune responses, we used ELISPOT to quantify the number of in vivoprimed capsid and transgene Th1 (IFNγ) responses (FIG. 2B). Elevatedcapsid and transgene reactive Th1 responses were observed in WT but notTLR9KO vector recipients indicating an abrogation of Th1 responses inthe absence of TLR9 signaling. These findings demonstrate the inhibitoryeffect of TLR9 signaling blockade on T cell effector function followingstimulation with AAVrh32.33 capsid and transgene antigens.

D. Minimal Cellular Infiltrate Observed in Muscle FollowingAAVrh32.33nLacZ I.M. Injection Observed in TLR9 Deficient Mice

Heavy CD4+ and CD8+ cellular infiltrate has been observed in WT C57BL/6mice following AAVrh32.33 intramuscular vector transduction [Mays et al,J Immunol, 2009, cited above]. To determine the requirement for TLR9signaling in the induction of this extensive infiltrate, WT and TLR9KOmuscle sections were stained with anti-CD4 and anti-CD8 Ab and examinedby fluorescent microscopy at 35 and 60 days vector post-administration(FIG. 3). As expected, significant cellular infiltrate was detected inWT mice. In contrast, minimal cellular infiltrate was observed in TLR9KOmice. These results are consistent with a TLR9 dependent mechanism ofcellular infiltrate in response to intramuscular AAVrh32.33nLacZtransduction.

E. Enhanced Transgene Expression Observed in the Muscles of TLR9Deficient Mice Injected with AAV8

Historically, AAV8 gene delivery to WT C57BL/6 muscle tissue results inminimal Th1 responses, negligible cellular infiltrate and prolongedtransgene expression [Mays et al, J Immunol, 2009, cited above]. Toinvestigate the role of TLR9 detection of AAV8 and its effect, if any,on AAV8 gene expression, WT and TLR9KO mice were injectedintramuscularly with 1×10¹¹ GC of AAV8nLacZ. X-gal histochemical stainof muscle sections 35 and 60 days post vector administration (FIG. 4).Enhanced expression is detected in the muscle of TLR9KO mice indicatingthe detection of AAV8 through this innate immune sensor and an effect ontransgene expression even in the case of an AAV vector that induces aminimal immune response.

F. TLR9 Signaling is Both Necessary and Sufficient to Upregulate MHC IIExpression on a Muscle Tissue

Muscle tissue possesses a unique function as a non-professional antigenpresenting cell (APC) that can effectively stimulate both CD4+ and CD8+T cells to survive, proliferate, and acquire effector function. Toassess the ability of AAVrh32.33 to induce MHC II expression on skeletalmuscle, gastroc sections from WT and TLR9KO mice that received I.M.injection of 1×10¹¹ GC of AAV8rh32.33nLacZ or AAV8nLacZ, were stainedwith an anti-MHC II Ab and examined by fluorescent microscopy 35 dayspost gene transfer (FIG. 5). WT muscle tissue transduced withAAVrh32.33nLacZ revealed significant upregulation of MHC II on skeletalmuscle, which was not present on muscle tissue from TLR9 deficient miceadministered AAVrh32.33nLacZ or WT mice that received AAV8. Thesefindings reveal the requirement of TLR9 signaling for MHC II expressionon muscle following AAVrh32.33, while AAV8 minimally induces MHC IIexpression.

G. AAVrh32.33 Induces Early Innate Immune Gene Transcript Induction inWT, but not TLR9, Deficient Mice

Zaiss et al. demonstrated that adenovirus vector transduction of humanHeLa cells and murine renal epithelium-derived cells induce theexpression of numerous chemokines and inflammatory cytokines [Zaiss, A.,et al,. 2002. J of Virol 76 (9) 4580-4590]. To assay for innate immunegene transcript induction following intramuscular injection ofAAVrh32.33 and AAV8 in WT or TLR9 deficient mice, we performedquantitative RT-PCR at early kinetic time points to detect innatechemokine and inflammatory transcript levels following gene transfer(FIG. 6). Transcript levels of MIP-1α, MIP-1β, MIP-2, MCP-1, IL-1α andIL-6 were quantified and expressed as fold induction over mock treated(PBS injected) WT mice. A dramatic induction of both chemokine andcytokine transcripts was observed in WT mice transduced with AAVrh32.33,but not in TLR9KO mice, or in WT mice administered AAV8. Collectively,our data suggests that TLR9 signaling is both necessary and sufficientto induce innate (FIG. 6) and adaptive (FIG. 2) immune responses towardan immunogenic AAV vector. Our observation that TLR9 deficientAAVrh32.33 vector recipients exhibit minimal immunoreactivity and stabletransgene expression led us to hypothesize that CpG depleted AAVrh32.33vectors would escape immune detection and exhibit long-term transgeneexpression.

H. CpG Depleted Vector Generation

TLR9 acts as an innate immune sensor that specifically recognizes andresponds to unmethylated CpG motifs present in ˜7% of microbial genomescompared to ˜1% vertebrate DNA. Systemic delivery of cationiclipid-plasmid DNA (pDNA) vectors that contain CpG motifs stimulate acuteinflammatory responses with adverse effects on transgene expression[Yew, N. S., et al, 2002, Mol Ther 5(6): 731-738]. CpG depleted plasmidDNA vectors, on the other hand, exhibit long-term expression andenhanced safety. To determine whether CpG depleted AAVrh32.33 vectorswould abrogate the robust cellular immune response and transgene loss weobserved in the previous experiments (FIG. 1-3, 5, 6), we generated aLacZ CpG depleted vector genome that retained a mere 16 CpGs located inthe inverted terminal repeat sequence (FIGS. 7A and 7B; SEQ ID NO: 4 and5). See, also, sequences provided below. CpGs in the EF1-α promoter, CMVenhancer, LacZ transgene, intron, and SV40 polyA were reduced in the CpGdepleted vector (CpG−), while the WT vector (CpG+), which contained thesame backbone sequence, retained 16 CpGs in the ITRs as well as 308 CpGsin the LacZ transgene (total=324 CpGs). The sequence of the CpG-modifiedLacZ transgene is provided in FIG. 11 [SEQ ID NO: 7].

AAV2 ITR alignment document SEQ ID NO: 1: AAV2 ITRSEQ ID NO: 2: CpG depleted ITR SEQ ID NO: 3: Consensus ITR

I. CpG Depleted AAV2/Rh32.33 Vectors Exhibit Stable Transgene Expression

To test our hypothesis that CpG depleted AAVrh32.33 vectors wouldexhibit prolonged transgene expression, X-gal histochemical stain ofgastroc tissues from WT mice injected I.M. with 1E11 GC ofAAVrh32.33LacZCpG+ or AAVrh32.33LacZCpG− were assessed (FIG. 8). Asexpected, X-gal stain of WT AAVrh32.33LacZCpG+ transduced muscleexhibited a steady loss of detectable βgal expression. Conversely, themuscle sections from CpG depleted AAVrh32.33LacZ transduced micedisplayed robust and stable transgene expression. Hence, the steady lossof LacZ transgene expression following AAVrh32.33LacZ gene transfer isdependent on vector genome CpG motifs.

J. Evidence of Hypertrophy in AAVrh32.33LacZCpG+Transduced Muscle

Acute and chronic inflammatory responses are strongly implicated in theinduction of a pro-fibrotic environment [Faust, S. M., et al, 2009, J ofImmunol 183: 7297-7803]. This inflammatory response stimulates collagendeposition and is commonly associated with the development of cellularhypertrophy. To investigate the impact of TLR9 signaling and thedevelopment of hypertrophy following intramuscular AAV gene transfer,gastroc tissue from AAVrh32.33LacZCpG+ or CpG− vector transduced micewas weighed and compared to total body weight (FIG. 9). A statisticallysignificant increase in muscle weight was observed in AAAVrh32.33CpG+compared to CpG depleted vector transduced mice. These data reveal theassociation of AAVrh32.33CpG+ gene transfer and TLR9 stimulatedinflammation in the development of muscle hypertrophy.

K. CpG Depletion Significantly Reduces the Percentage of LacZ ReactiveCD8+ T Cells and T Cell Effector Function

Transgene stability observed in the AAVrh32.33LacZCpG− transduced musclesections (FIG. 8) strongly suggests an abrogated adaptive immuneresponse toward transgene and capsid antigen. To assess the requirementfor CpG motifs in the induction of an adaptive immune response towardAAVrh32.33, MHC I tetramer stain and ELISPOT assays were used toquantify transgene reactive CD8+ T cells and primed transgene and capsidresponsive IFNγ-producing cells as described above (FIG. 9). Mice thatreceived the CpG depleted AAVrh32.33LacZ vector exhibited a significantreduction in the percentage of LacZ responsive CD8+ T cell populationcompared to control mice (FIG. 9A). Furthermore, a significant decreasein primed transgene and capsid antigen reactive Th1 responses wereobserved in mice that received the AAVrh32.33CpG− but not CpG+ vector(9B). These findings indicate the ability of a CpG depleted vector toescape immunoreactivity following gene transfer.

L. CpG Depleted AAVrh32.33LacZ Vector Gene Transfer Corresponds withMinimal Cellular Infiltrate and MHC II Expression

Minimal cellular infiltrate and MHC II expression was revealed in musclesections in the absence of TLR9 signaling following AAVrh32.33nLacZ genetransfer (FIGS. 3 and 5). These data are consistent with a TLR9dependent mechanism of cellular infiltrate and MHC II skeletal musclegene induction in response to the immunogenic AAV vector. If TLR9signaling is both necessary and significant for these phenomenon, it isreasonable to suggest that CpG depleted AAVrh32.33LacZ vectors shouldexhibit similar histological findings to TLR9KO mice. To test thishypothesis, 1×10¹¹ GC of AAVrh32.33LacZCpG+ and CpG− vectors wereinjected intramuscularly and muscle sections were stained with anti-CD4,anti-CD8 and anti-MHC II Ab (FIG. 10). Consistent with our theory,muscle transduced with AAVrh32.33CpG-vector revealed minimal cellularinfiltrate and MHC II expression compared to AAVrh32.33CpG+ transducedmuscle. These data reveal the ability of CpG depleted AAV vectors toestablish long-term transgene expression (FIG. 8), evade immuneactivation (FIG. 9), prevent the infiltration of effector T cells, andsubvert the induction of skeletal muscle MHC II expression (FIG. 10).

Example 2

To measure LacZ expression of RhCpG+ and CpG− constructs, HeLa cellswere transfected with CpG+ and CpG− AAV expression plasmids. Four dayspost transfection cells were assayed for β-galactosidase activity usingthe Mammalian β-galactosidase assay kit as instructed for adherentcells. Absorbance was measured at 405 nm on a TECAN Infinite M1000 PROplate reader. CpG+ and CpG− AAV vector constructs exhibited comparableLacZ plasmid expression. These data demonstrate that transgene loss inthe skeletal muscle of RhCpG+ gene transferred is not due todifferential β-gal expression levels at the plasmid level.

To assess transgene stability and cellular infiltrate at an earlykinetic time point, mice were injected intramuscularly with 1×10¹¹ GC ofRhCpG+ or RhCpG− vector. 14 days post vector injection, gastrocnemiuswas harvested and skeletal muscle cryosections were stained with CD4 orCD8 monoclonal antibody (MAb) as well as X-gal. Stable transgeneexpression and minimal cellular infiltrate was observed in animals thatreceive RhCpG− vector. This indicates that reduced cellular immunity isobserved as early as day 14 post vector transduction.

To examine adaptive immunity toward AAV associated antigen, splenocyteswere harvested 7 and 14 days post intramuscular injection of RhCpG+ orRhCpG−. ELISPOT analysis was performed to quantify spots of IFNγ permillion cells. Similar Th1 responses were observed at the early timepoint. In contrast, a robust Th1 response toward transgene antigen isobserved only in the RhCpG+ vector transduced animals at day 14. Thesedata demonstrate suppressed Th1 responses following RhCpG−administration at an early kinetic time point.

To determine whether tolerance is induced toward the β-gal transgenefollowing RhCpG− gene transfer, mice were injected with 1×10¹¹ GC ofRhCpG+ or RhCpG− in the right gastrocnemius muscle. 60 days post primaryadministration, mice were injected in the contralateral muscle with1×10¹¹ GC of RhCpG+. Both right and left gastrocnemius tissue washarvested and muscle cryosections were stained with CD4/CD8 MAb as wellas X-gal 35 days post-secondary injection. Transgene expression andminimal cellular infiltrate was exhibited in the right gastrocnemius ofthe RhCpG− gene transferred mice indicating a localized tolerance in theskeletal muscle even following an immunogenic vector administration.

To analyze the Th1 response in the animals that receive a secondaryinjection of RhCpG+, mice were injected with RhCpG+ or RhCpG− in theright gastrocnemius muscle and 60 days post primary administration,injected in the contralateral muscle with RhCpG+(FIG. 17). Splenocyteswere harvested 35 days post-secondary administrations and ELISPOT wasperformed to quantify spots of IFNγ per million cells. Interestingly,comparable Th1 responses in mice gene transferred initially with RhCpG+or RhCpG− vector and readministered RhCpG+ vector, even in the presenceof localized tolerance in the skeletal muscle of RhCpG− transducedanimals. These data suggests that even in the presence of transgenereactive T cells, a factor localized in the skeletal muscle of miceadministered the CpG depleted vector prevents the extinguishment ofβ-gal expression.

All publications cited in this specification are incorporated herein byreference, is U.S. Patent Application No. 61/785,368, filed Mar. 14,2013. The sequence listing filed herewith, file “UPN_Y6335US_ST25.txt”,is hereby incorporated by reference. While the invention has beendescribed with reference to a particularly preferred embodiment, it willbe appreciated that modifications can be made without departing from thespirit of the invention. Such modifications are intended to fall withinthe scope of the appended claims.

1. A recombinant adeno-associated viral (AAV) vector comprising AAVcapsid having packaged therein a nucleic acid molecule comprising atleast one copy of AAV inverted terminal repeat (ITR) sequences and anexogenous gene sequence under the control of regulatory sequences whichcontrol expression of the gene product, wherein said sequences of saidnucleic acid molecule are modified to reduce CpG di-nucleotides suchthat in immune response to the vector is reduced as compared to theunmodified AAV vector.
 2. The recombinant AAV vector according to claim1, wherein the vector is deleted of functional AAV capsid codingsequences and functional AAV rep coding sequences.
 3. The recombinantAAV vector according to claim 1, wherein the exogenous gene sequencecontains a reduced number of CpG di-nucleotides as compared to thenative coding sequence and the untranslated regions for the geneproduct.
 4. The recombinant AAV vector according to claim 1, wherein theregulatory sequences are mutated to reduce or eliminate CpGdi-nucleotides.
 5. The recombinant AAV vector according to claim 1,wherein the regulatory sequences are selected from the group consistingof one or more of: a promoter, an enhancer, an intron, microRNAs, andpolyA sequence.
 6. The recombinant AAV vector according to claim 1,wherein the one or both of the 5′ and the 3′ AAV ITRs are mutated toreduce or eliminate native CpG di-nucleotides.
 7. The recombinant AAVvector according to claim 1 comprising a 5′ AAV ITRs selected from thegroup consisting of a CpG-depleted AAV ITR, a wild-type AAV ITR, aCpG-modified self-complementary ITRs, and a self-complementary ITR. 8.The recombinant AAV vector according to claim 1 comprising a 3′ AAV ITRsselected from the group consisting of a CpG-depleted AAV ITR, awild-type AAV ITR, a CpG-modified self-complementary ITRs, and aself-complementary ITR.
 9. The recombinant AAV vector according to claim1, wherein untranslated regions and/or any vector DNA sequences areCpG-depleted.
 10. The recombinant AAV vector according to claim 1,wherein the number of CpG di-nucleotides in the nucleic acid molecule isreduced by at least 30% as compared to a nucleic acid molecule havingthe corresponding native ITRs, native exogenous gene sequence and nativeregulatory sequence.
 11. The recombinant AAV vector according to claim1, wherein the AAV vector retains at least 95% of the protein expressionlevels as compared to a nucleic acid molecule having the correspondingnative ITRs, native exogenous gene sequence and native regulatorysequence.
 12. The recombinant AAV vector according to claim 1, whereinthe AAV vector is completely depleted of CpG di-nucleotides.
 13. Therecombinant AAV vector according to claim 1, wherein said AAV vector hasa capsid selected from AAV8 and rh32/33.
 14. A pharmaceuticalcomposition comprising a recombinant AAV vector according claim 1 and apharmaceutically acceptable carrier.
 15. The composition according toclaim 14, wherein said composition is formulated for parenteraldelivery.
 16. The composition according to claim 14, wherein saidcomposition is formulated for intramuscular delivery.
 17. A method forimproving adeno-associated virus (AAV)-mediated gene expression, saidmethod comprising: a) generating an AAV viral particle comprising amodified packaging insert, wherein said packaging insert comprises anucleic acid molecule comprising two AAV inverted terminal repeat (ITR)and an exogenous gene sequence under the control of regulatory sequenceswhich control expression of the gene product, wherein said sequences ofsaid nucleic acid molecule are modified to reduce CpG di-nucleotidessuch that in immune response to the vector is reduced as compared to theunmodified AAV vector without significant reduction in expression of thegene product; and b) delivering the AAV to a subject.
 18. The methodaccording to claim 17, wherein the AAV ITRs are mutated to eliminatenative CpG di-nucleotides.
 19. The method according to claim 17, whereinthe regulatory sequences are mutated to eliminate CpG di-nucleotides.20. The method according to claim 17, wherein the exogenous genesequence contains a reduced number of CpG di-nucleotides as compared tothe native coding sequence for the gene product.
 21. A regimen forrepeat administration of a gene product, said regimen comprising: a)delivering to a subject an adeno-associated viral (AAV) vector having anAAV capsid having packaged therein a nucleic acid molecule comprisingtwo AAV inverted terminal repeats (ITR) and an exogenous gene sequenceunder the control of regulatory sequences which control expression ofthe gene product, wherein said sequences of said nucleic acid moleculeare modified to reduce CpG di-nucleotides such that in immune responseto the vector is reduced as compared to the unmodified AAV vectorwithout significant reduction in expression of the gene product; and b)delivering to the subject a second vector comprising the exogenous genesequence.